Cell-free production of sugars

ABSTRACT

Provided herein, in some embodiments, are systems, methods, and compositions (e.g., cells and cell lysates) for enzymatically converting a polymeric glucose carbohydrate (e.g., starch) to sugar.

RELATED APPLICATIONS

This application is a continuation application of of internationalapplication number PCT/US2018/012516, filed Jan. 5, 2018, which claimspriority under 35 U.S.C. § 119(e) to U.S. provisional applications U.S.Ser. No. 62/443,447, filed Jan. 6, 2017, and U.S. Ser. No. 62/538,181,filed Jul. 28, 2017, each of which is incorporated herein by referencein its entirety.

BACKGROUND

Existing technologies for the conversion of starch to simple sugarsemploy multiple biotransformation reactions, with extensive purificationprocesses following each biotransformation. While the biotransformationprocesses are relatively inexpensive, owing to the application ofimmobilized enzymes and continuous production systems, the downstreamprocessing impacts cost dramatically.

SUMMARY

Provided herein are cell free systems, methods, compositions and kitsfor the enzymatic conversion of polymeric glucose, such as starch (e.g.,amylose and/or amylopectin), glycogen, or any partially hydrolyzedderivative thereof such as maltodextrin, or cellodextrin (which may beused interchangeably with the term cellulose) to pentose (e.g., ribose,arabinose, or xylulose) or hexose (e.g., allulose, glucose, or fructose)sugars. The methods of the present disclosure implement sugar productionpathways in cell-free reactions (e.g., a one-pot (single) cell-freereaction), to convert starch and/or cellulose/cellodextrin to hexoseand/or pentose sugars. Unlike processes that typically involvephosphorylation of substrates such as glucose to glucose 6-phosphate andemploy high-energy phosphate sources such as ATP andphosphoenoylpyruvate, the processes described herein typically replacehigh energy phosphate sources with, for example, inexpensive inorganicphosphate (P_(i)). In some embodiments, an α-glucan phosphorylase (alsoreferred to as a starch phosphorylase) (EC 2.4.1.1) is used to convertstarch to glucose 1-phosphate, which is then converted to glucose6-phosphate via a phosphoglucomutase (EC 5.4.2.2, 5.4.2.5, or 5.4.2.6).In other embodiments, a cellodextrin phosphorylase (also referred to ascellulose phosphorylase or β-(1-4) glucan phosphorylase) (EC 2.4.1.49)is used to convert cellulose/cellodextrin to glucose 1-phosphate, whichis then converted to glucose 6-phosphate via a phosphoglucomutase (EC5.4.2.2, 5.4.2.5, or 5.4.2.6). Subsequent enzymatic reaction(s) of aparticular sugar production pathway as provided herein are largelyproduct specific. A sugar phosphatase (EC 3.1.3.-) is used to convertthe final product. Thus, the reaction thermodynamics—phosphorylation ofthe substrate to desphosphorylation of the product—favor the product.

Further, the enzymatic conversion reactions described herein areessentially irreversible, thus supporting high yields of the desiredhexose and pentose sugars. By contrast, typical biotransformationmethods for converting starch or cellulose/cellodextrin to allulose, forexample, employ three distinct processes, two of which are reversible,with the final concentration of the product being governed by thethermodynamics of the enzymes being utilized. Starch, for example, isconverted to glucose, glucose is isomerized to fructose, and fructose isepimerized to allulose. The isomerization of glucose to fructose has ayield of approximately 45%, thus significant downstream processing isrequired to yield a pure product and recycle uncatalyzed substrate.Similarly, the epimerization of fructose to allulose has a yield of˜20%, again requiring substantial downstream processing to yield apurified product and recycle uncatalyzed substrate. The ability todirectly transform starch to the product of interest in the cell-freesystems described herein reduces cost by reducing downstream processingand the loss of substrate.

Advantageously, many of the enzymes used in the processes providedherein are thermostable, which (1) enables thermal inactivation ofdeleterious activities contained within cellular lysates in which theconversion process is performed, and (2) decreases the chances ofmicrobial contamination negatively impacting production runs. Theenzymes of these conversion pathways can be isolated from thermophilic,mesophilic, or psychrophilic organisms and/or, in some embodiments, canbe engineered to increase (or decrease) the thermostability of theenzymes. A thermophilic organism (thermophile) thrives at hightemperatures, between 41° C. and 122° C. (106° F. and 252° F.). Amesophilic organism (mesophile) thrives at moderate temperatures,between 20° C. and 45° C. (68° F. and 113° F.). A psychrophilic organism(psychrophile) thrives at cold temperatures, between −20° C. and 10° C.(−4° F. and 50° F.).

Thus, some aspects of the present disclosure provide methods forproducing a sugar (e.g., allulose, glucose, fructose, sorbitol,ribulose, ribose, and/or arabinose), the method comprising (a) culturingat least two cell populations, wherein cells of each population areengineered to express at least one thermostable enzyme of a sugarproduction pathway described herein to produce at least two culturedpopulations of cells expressing different enzymes, (b) lysing cells ofthe at least two cultured populations to produce at least two celllysates, (c) combining the at least two cell lysates to produce a celllysate mixture that comprises thermostable enzymes of the sugarproduction pathway, (d) heating the cell lysate mixture to a temperaturethat inactivates undesired native enzymatic activities but does notinactivate the thermostable enzymes of step (c) to produce aheat-inactivated lysate, and (e) incubating the reaction mixture in thepresence of a substrate (e.g., starch, glycogen, or any partiallyhydrolyzed derivative thereof) and a phosphate source (e.g., inorganicphosphate) to produce the sugar.

In some embodiments, a cell-free method for producing a sugar (e.g.,allulose, glucose, fructose, sorbitol, ribulose, ribose, and/orarabinose) comprises (a) culturing at least two cell populations,wherein cells of each population are engineered to express at least oneenzyme of a sugar production pathway described herein to produce atleast two cultured populations of cells expressing different enzymeswherein at least one of the enzymes of the sugar production pathway isthermostable, (b) lysing cells of the at least two cultured populationsto produce at least two cell lysates, (c) optionally heating one or moreof the cell lysates of step (b) to a temperature that inactivatesundesired native enzymatic activities but does not inactivate thethermostable enzyme(s) of step (a) to produce a heat-inactivated lysate,(d) combining the cell lysates of step (b) and (c) to produce a celllysate mixture that comprises the enzymes of the sugar productionpathway, wherein at least one of the foregoing enzymes is thermostable,and (e) incubating the reaction mixture in the presence of a substrate(e.g., starch, glycogen, or any partially hydrolyzed derivative thereof)and a phosphate source (e.g., inorganic phosphate) to produce the sugar.

In some embodiments, a cell-free method for producing a sugar (e.g.,allulose, glucose, fructose, sorbitol, ribulose, ribose, and/orarabinose) comprises (a) culturing at least two cell populations,wherein cells of each population are engineered to express at least onethermostable enzyme of a sugar production pathway described herein toproduce at least two cultured populations of cells expressing differentenzymes, (b) lysing cells of the at least two cultured populations toproduce at least two cell lysates, (c) combining the at least two celllysates to produce a cell lysate mixture, (d) heating the cell lysatemixture to a temperature that inactivates undesired native enzymaticactivities but does not inactivate the thermostable enzymes of step (a)to produce a heat-inactivated lysate, (e) adding to the heat-inactivatedlysate at least one purified enzyme of the sugar production pathway, and(f) incubating the reaction mixture in the presence of a substrate(e.g., starch, glycogen, or any partially hydrolyzed derivative thereof)and a phosphate source (e.g., inorganic phosphate) to produce the sugar.

In some embodiments, a cell-free method for producing a sugar (e.g.,allulose, glucose, fructose, sorbitol, ribulose, ribose, and/orarabinose) comprises (a) culturing at least two cell populations,wherein cells of each population are engineered to express at least oneenzyme of a sugar production pathway described herein to produce atleast two cultured populations of cells expressing different enzymes,wherein at least one of the foregoing enzymes is thermostable, (b)lysing cells of the at least two cultured populations to produce atleast two cell lysates, (c) optionally heating one or more of the celllysates of step (b) to a temperature that inactivates undesired nativeenzymatic activities but does not inactivate the thermostable enzymes ofstep (a) to produce a heat-inactivated lysate, (d) combining the celllysates of step (b) and (c) to produce a cell lysate mixture, (e) addingto the cell lysate mixture at least one purified enzyme of the sugarproduction pathway, and (f) incubating the reaction mixture in thepresence of a substrate (e.g., starch, glycogen, or any partiallyhydrolyzed derivative thereof) and a phosphate source (e.g., inorganicphosphate) to produce the sugar.

Some aspects of the present disclosure provide cell-free methods forproducing allulose, the methods comprising (a) culturing cellsengineered to express a α-glucan phosphorylase (also referred to as astarch phosphorylase), a phosphoglucomutase, a phosphoglucoisomerase, anallulose 6-phosphate epimerase, and an allulose 6-phosphate phosphataseto produce cultured cells that express the enzymes, wherein at least oneof the foregoing enzymes is thermostable, (b) lysing the cultured cellsto produce a cell lysate, (c) heating the cell lysate to a temperaturethat inactivates native enzymatic activity but does not inactivate thethermostable enzyme(s) of step (a) to produce a heat-inactivated lysate,and (d) incubating the heat-inactivated lysate in the presence ofstarch, glycogen, or any partially hydrolyzed derivative thereof and aphosphate source (e.g., inorganic phosphate) to produce allulose. Insome embodiments, at least one purified enzyme is added to the celllysate before or after step (c). It should be understood that the cellsmay be lysed by any means, including mechanical, chemical, enzymatic,osmotic or thermal lysis. Thus, the lysing step and the heating (heatinactivation) step may be combined as a single step of heating the cellsto a temperature that lyses the cells and inactivates native enzymaticactivity.

In some embodiments, the cell-free methods comprise (a) culturing atleast two cell populations, wherein cells of each population areengineered to express at least one enzyme selected from the groupconsisting of α-glucan phosphorylases, phosphoglucomutases,phosphoglucoisomerases, allulose 6-phosphate epimerases, and allulose6-phosphate phosphatases to produce at least two cultured populations ofcells expressing different enzymes, wherein at least one of foregoingenzymes is thermostable, (b) lysing cells of the at least two culturedpopulations to produce at least two cell lysates, (c) combining the atleast two cell lysates to produce a cell lysate mixture that comprises aα-glucan phosphorylase, a phosphoglucomutase, a phosphoglucoisomerase,an allulose 6-phosphate epimerase, and an allulose 6-phosphatephosphatase, (d) heating the cell lysate mixture to a temperature thatinactivates native enzymatic activity but does not inactivate thethermostable enzyme(s) of step (c) to produce a heat-inactivated lysate,and (e) incubating the reaction mixture in the presence of a starch,glycogen, or any partially hydrolyzed derivative thereof and a phosphatesource (e.g., inorganic phosphate) to produce allulose.

In other embodiments, the cell-free methods comprise (a) culturing atleast two cell populations, wherein cells of each population areengineered to express at least one enzyme selected from the groupconsisting of α-glucan phosphorylases, phosphoglucomutases,phosphoglucoisomerases, allulose 6-phosphate epimerases, and allulose6-phosphate phosphatases to produce at least two cultured populations ofcells expressing different enzymes, wherein at least one of theforegoing enzymes is thermostable, (b) lysing cells of the at least twocultured populations to produce at least two cell lysates, (c) combiningthe at least two cell lysates to produce a cell lysate mixture (d)heating the cell lysate mixture to a temperature that inactivates nativeenzymatic activity but does not inactivate the thermostable enzyme(s) ofstep (a) to produce a heat-inactivated lysate, (e) adding to theheat-inactivated lysate at least one purified enzyme selected from thegroup consisting of α-glucan phosphorylases, phosphoglucomutases,phosphoglucoisomerases, allulose 6-phosphate epimerases, and allulose6-phosphate phosphatases to produce a reaction mixture comprising aα-glucan phosphorylase, a phosphoglucomutase, a phosphoglucoisomerase,an allulose 6-phosphate epimerase, and an allulose 6-phosphatephosphatase, and (f) incubating the reaction mixture in the presence ofa starch, glycogen, or any partially hydrolyzed derivative thereof and aphosphate source (e.g., inorganic phosphate) to produce allulose.

In some aspects of the present disclosure, it may be preferable to usecellulose/cellodextrin as a starting substrate. Thus, some aspects ofthe present disclosure provide cell-free methods for producing allulose,the methods comprising (a) culturing cells engineered to express acellodextrin phosphorylase, a phosphoglucomutase, aphosphoglucoisomerase, an allulose 6-phosphate epimerase, and anallulose 6-phosphate phosphatase to produce cultured cells that expressthe enzymes, wherein at least one of the foregoing enzymes isthermostable, (b) lysing the cultured cells to produce a cell lysate,(c) heating the cell lysate to a temperature that inactivates nativeenzymatic activity but does not inactivate the thermostable enzyme(s) ofstep (a) to produce a heat-inactivated lysate, and (d) incubating theheat-inactivated lysate in the presence of cellodextrin and a phosphatesource (e.g., inorganic phosphate) to produce allulose.

In some embodiments, the cell-free methods comprise (a) culturing atleast two cell populations, wherein cells of each population areengineered to express at least one enzyme selected from the groupconsisting of cellodextrin phosphorylases, phosphoglucomutases,phosphoglucoisomerases, allulose 6-phosphate epimerases, and allulose6-phosphate phosphatases to produce at least two cultured populations ofcells expressing different enzymes, wherein at least one of foregoingenzymes is thermostable, (b) lysing cells of the at least two culturedpopulations to produce at least two cell lysates, (c) combining the atleast two cell lysates to produce a cell lysate mixture that comprises acellodextrin phosphorylase, a phosphoglucomutase, aphosphoglucoisomerase, an allulose 6-phosphate epimerase, and anallulose 6-phosphate phosphatase, (d) heating the cell lysate mixture toa temperature that inactivates native enzymatic activity but does notinactivate the thermostable enzyme(s) of step (c) to produce aheat-inactivated lysate, and (e) incubating the reaction mixture in thepresence of a cellodextrin and a phosphate source (e.g., inorganicphosphate) to produce allulose.

In other embodiments, the cell-free methods comprise (a) culturing atleast two cell populations, wherein cells of each population areengineered to express at least one enzyme selected from the groupconsisting of cellodextrin phosphorylases, phosphoglucomutases,phosphoglucoisomerases, allulose 6-phosphate epimerases, and allulose6-phosphate phosphatases to produce at least two cultured populations ofcells expressing different enzymes, wherein at least one of theforegoing enzymes is thermostable, (b) lysing cells of the at least twocultured populations to produce at least two cell lysates, (c) combiningthe at least two cell lysates to produce a cell lysate mixture (d)heating the cell lysate mixture to a temperature that inactivates nativeenzymatic activity but does not inactivate the thermostable enzyme(s) ofstep (a) to produce a heat-inactivated lysate, (e) adding to theheat-inactivated lysate at least one purified enzyme selected from thegroup consisting of cellodextrin phosphorylases, phosphoglucomutases,phosphoglucoisomerases, allulose 6-phosphate epimerases, and allulose6-phosphate phosphatases to produce a reaction mixture comprising acellodextrin phosphorylase, a phosphoglucomutase, aphosphoglucoisomerase, an allulose 6-phosphate epimerase, and anallulose 6-phosphate phosphatase, and (f) incubating the reactionmixture in the presence of a cellodextrin and a phosphate source (e.g.,inorganic phosphate) to produce allulose.

The cell-free production of other sugars, such as glucose, fructose,mannose, sorbitol, ribulose, ribose, and arabinose is also encompassedby the present disclosure.

In some embodiments, the presented sugar pathways require the balancingof energetic cofactors, such as NADH, NADPH, NAD⁺, or NADP⁺. This can bedone through cofactor regeneration systems. In these instances, NADH andNADPH are referred to as “reduced cofactors” or “reducing agents,” andNAD⁺ and NADP⁺ are referred to as “oxidized cofactors” or “oxidizingagents.” For instances with excess reducing agents, an NAD(P)H oxidase(EC#1.6.3.1, 1.6.3.2, 1.6.2.3, or 1.6.3.4), can be used to burn excessreduced cofactors producing either H₂O₂, O⁻ ₂, or H₂O, depending on thetype of oxidase. When producing H₂O₂ and O₂ ⁻, which can causedetrimental damage to the lysate, superoxide dismutase (EC#1.15.1.1)and/or catalase (EC#1.11.1.6) can be used in tandem to convert theharmful species to H₂O and O₂. For instances with excess oxidizingagents, a cofactor regeneration system can be used to reduce theoxidized cofactors back to their reduced forms. Some examples includethe use of formate dehydrogenase (EC#1.2.1.2) to oxidize formate to CO₂while reducing NAD(P)⁺ to NAD(P)H, or the use of phosphonatedehydrogenase (EC#1.20.1.1) or sulfite oxidoreductase (EC#1.8.1.2) tooxidize the respective inorganic salts to phosphate and sulfate,resulting in reduced NAD(P)H.

Also provided herein are engineered cells, cell lysates, and reactionmixtures comprising enzymes, such as thermostable enzymes, used for theproduction of a particular sugar of interest (e.g., allulose, glucose,fructose, sorbitol, ribulose, ribose, and/or arabinose).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of an enzymatic pathway for the conversion ofstarch to allulose. The meaning of the abbreviations is as follows:G1P=glucose 1-phosphate, G6P=glucose 6-phosphate, F6P=fructose6-phosphate, A6P=allulose 6-phosphate, and PO₄=inorganic phosphate.

FIG. 2 is a schematic of two enzymatic pathways for the conversion ofstarch to glucose. The meaning of the abbreviations is as follows:G1P=glucose 1-phosphate, G6P=glucose 6-phosphate, and PO₄=inorganicphosphate.

FIG. 3 is a schematic of two enzymatic pathways for the conversion ofstarch to fructose. The meaning of the abbreviations is as follows:G1P=glucose 1-phosphate, G6P=glucose 6-phosphate, F6P=fructose6-phosphate, and PO₄=inorganic phosphate.

FIG. 4 is a schematic of an enzymatic pathway for the conversion ofstarch to sorbitol. The meaning of the abbreviations is as follows:G1P=glucose 1-phosphate, G6P=glucose 6-phosphate,S6P=sorbitol-6-phosphate, F6P=fructose 6-phosphate, NADPH=nicotinamideadenine dinucleotide phosphate (reduced form), NADP⁺=nicotinamideadenine dinucleotide phosphate, and PO₄=inorganic phosphate.

FIG. 5 is a schematic of an enzymatic pathway for the conversion ofstarch to ribulose. The meaning of the abbreviations is as follows:G1P=glucose 1-phosphate, G6P=glucose 6-phosphate,6PGL=6-phosphogluconolactone, 6PG=6-phosphogluconate, Ru5P=ribulose5-phosphate, NADPH=nicotinamide adenine dinucleotide phosphate (reducedform), NADP⁺=nicotinamide adenine dinucleotide phosphate, CO₂=carbondioxide, and PO₄=inorganic phosphate.

FIG. 6 is a schematic of an enzymatic pathway for the conversion ofstarch to ribose. The meaning of the abbreviations is as follows:G1P=glucose 1-phosphate, G6P=glucose 6-phosphate,6PGL=6-phosphogluconolactone, 6PG=6-phosphogluconate, Ru5P=ribulose5-phosphate, R5P=ribose 5-phosphate, NADPH=nicotinamide adeninedinucleotide phosphate (reduced form), NADP⁺=nicotinamide adeninedinucleotide phosphate, CO₂=carbon dioxide, and PO₄=inorganic phosphate.

FIG. 7 is a schematic of an enzymatic pathway for the conversion ofstarch to arabinose. The meaning of the abbreviations is as follows:G1P=glucose 1-phosphate, G6P=glucose 6-phosphate,6PGL=6-phosphogluconolactone, 6PG=6-phosphogluconate, Ru5P=ribulose5-phosphate, Ar5P=arabinose 5-phosphate, NADPH=nicotinamide adeninedinucleotide phosphate (reduced form), NADP⁺=nicotinamide adeninedinucleotide phosphate, CO₂=carbon dioxide, and PO₄=inorganic phosphate.

FIG. 8 is a schematic of an enzymatic pathway for the conversion ofstarch to mannose. The meaning of the abbreviations is as follows:G1P=glucose 1-phosphate, G6P=glucose 6-phosphate, F6P=fructose6-phosphate, M6P=mannose 6-phosphate, and PO4=inorganic phosphate.

DETAILED DESCRIPTION

Described herein are enzymatic pathways used for the conversion ofstarch (e.g., amylose or amylopectin) or cellulose/cellodextrin topentose (e.g., ribose, arabinose, or xylulose) and/or hexose (e.g.,allulose, glucose, or fructose) sugars. The enzymatic pathways utilizeat least one α-glucan phosphorylase (also referred to as a starchphosphorylase) (EC 2.4.1.1) or at least one cellodextrin phosphorylase(also referred to as cellulose phosphorylase) (EC 2.4.1.49), at leastone phosphoglucomutase (EC 5.4.2.2, 5.4.2.5, or 5.4.2.6) and any numberof isomerases, epimerases, and/or sugar phosphatases, depending on thefinal product. In some embodiments, the enzymes or a portion of theenzymes are thermostable. These thermostable enzymes can withstand theheating step of the sugar production process that inactivate deleteriousactivities contained within cellular lysates in which the conversionprocesses are performed. This heat inactivation step decreases thechances of microbial contamination negatively impacting production runs.

Thus, the present disclosure provides, in some embodiments,highly-efficient and cost-effective methods, compositions, and systemsfor producing sugars such as hexose and pentose sugars. Non-limitingexamples of sugar production pathways and pathway enzymes are providedin Table 1 below.

TABLE 1 Summary of Exemplary Pathway Enzymes Pathway Substrate Enzymesglucose α(1-4) or β(1-4) α- or β-(1-4) glucan phosphorylase (EC 2.4.1.1,2.4.1.49), production glucans phosphoglucomutase (EC 5.4.2.2, 5.4.2.5,5.4.2.6), glucose 6-phosphate phosphatase (EC 3.1.3.9, 3.1.3.58) glucoseα(1-4) or β(1-4) α- or β-(1-4) glucan phosphorylase (EC 2.4.1.1,2.4.1.49), production glucans glucose 1-phosphate phosphatase (EC3.1.3.10) fructose α(1-4) or β(1-4) α- or β-(1-4) glucan phosphorylase(EC 2.4.1.1, 2.4.1.49), production glucans phosphoglucomutase (EC5.4.2.2, 5.4.2.5, 5.4.2.6), phosphoglucoisomerase (EC 5.3.1.9), andfructose 6-phosphate phosphatase (EC 3.1.3.—, 3.1.3.58) allulose α(1-4)or β(1-4) α- or β-(1-4) glucan phosphorylase (EC 2.4.1.1, 2.4.1.49),production glucans phosphoglucomutase (EC 5.4.2.2, 5.4.2.5, 5.4.2.6),phosphoglucoisomerase (EC 5.3.1.9), allulose 6-phosphate epimerase (EC5.1.3.—), and allulose 6-phosphate phosphatase (EC 3.1.3.—, 3.1.3.58)sorbitol α(1-4) or β(1-4) α- or β-(1-4) glucan phosphorylase (EC2.4.1.1, 2.4.1.49), production glucans phosphoglucomutase (EC 5.4.2.2,5.4.2.5, 5.4.2.6), aldose dehydrogenase (EC 1.1.1.200), andsorbitol-6-phosphate phosphatase (EC 3.1.3.50, 3.1.3.58) sorbitol α(1-4)or β(1-4) α- or β-(1-4) glucan phosphorylase (EC 2.4.1.1, 2.4.1.49),production glucans phosphoglucomutase (EC 5.4.2.2, 5.4.2.5, 5.4.2.6),phosphoglucoisomerase (EC 5.3.1.9) sorbitol-6-phosphate 2-dehydrogenase(EC 1.1.1.140) sorbitol-6-phosphate phosphatase (EC 3.1.3.50, 3.1.3.58)ribulose α(1-4) or β(1-4) α- or β-(1-4) glucan phosphorylase (EC2.4.1.1, 2.4.1.49), production glucans phosphoglucomutase (EC 5.4.2.2,5.4.2.5, 5.4.2.6), glucose 6-phosphate dehydrogenase (EC 1.1.1.49,1.1.1.388, 1.1.1.363), 6-phosphogluconolactonase (EC 3.1.1.31),6-phosphogluconate dehydrogenase (EC 1.1.1.44, 1.1.1.343, 1.1.1.351),and ribulose 5-phosphate phosphatase (EC 3.1.3.—, 3.1.3.58) riboseα(1-4) or β(1-4) α- or β-(1-4) glucan phosphorylase (EC 2.4.1.1,2.4.1.49), production glucans phosphoglucomutase (EC 5.4.2.2, 5.4.2.5,5.4.2.6), glucose 6-phosphate dehydrogenase (EC 1.1.1.49, 1.1.1.388,1.1.1.363), 6-phosphogluconolactonase (EC 3.1.1.31), 6-phosphogluconatedehydrogenase (EC 1.1.1.44, 1.1.1.343, 1.1.1.351), and ribose5-phosphate isomerase (EC 5.3.1.6), and ribose 5-phosphate phosphatase(EC 3.1.3.—, 3.1.3.58) arabinose α(1-4) or β(1-4) α- or β-(1-4) glucanphosphorylase (EC 2.4.1.1, 2.4.1.49), production glucansphosphoglucomutase (EC 5.4.2.2, 5.4.2.5, 5.4.2.6), glucose 6-phosphatedehydrogenase (EC 1.1.1.49, 1.1.1.388, 1.1.1.363),6-phosphogluconolactonase (EC 3.1.1.31), 6-phosphogluconatedehydrogenase (EC 1.1.1.44, 1.1.1.343, 1.1.1.351), and arabinose5-phosphate isomerase (EC 5.3.1.13), and arabinose 5-phosphatephosphatase (EC 3.1.3.—, 3.1.3.58) mannose α(1-4) or β(1-4) α- orβ-(1-4) glucan phosphorylase (EC 2.4.1.1, 2.4.1.49), production glucansphosphoglucomutase (EC 5.4.2.2, 5.4.2.5, 5.4.2.6), phosphoglucoisomerase(EC 5.3.1.9), mannose 6-phosphate isomerase (EC 5.3.1.8), and mannose6-phosphate phosphatase (EC 3.1.3.—, 3.1.3.58)Allulose Production

Some aspects of the present disclosure provide methods, compositions,and systems for producing allulose. These methods, in some embodiments,include culturing cells engineered to express at least one α-glucanphosphorylase and/or at least one cellodextrin phosphorylase, at leastone phosphoglucomutase, at least one phosphoglucoisomerase, at least oneallulose 6-phosphate epimerase, at least one allulose 6-phosphatephosphatase, or a combination of at least two (e.g., at least three, orat least four) of the foregoing enzymes. In some embodiments, theα-glucan phosphorylase (and/or cellodextrin phosphorylase) and thephosphoglucomutase are expressed as a single fusion (chimeric) proteinor a bifunctional protein.

A fusion protein may be created by joining two or more gene or genesegments that code for separate proteins. Translation of this fusiongene results in a single or multiple polypeptides with functionalproperties derived from each of the original proteins. A polyfunctionalprotein is a single protein that has at least two different activities,wherein that functionality is a native biological function or the resultof an engineered enzyme fusion. Other enzymes may also be expressed as asingle fusion protein or a polyfunctional protein. Thus, a fusionprotein may contain multiple functionalities of any of the pathwayenzymes described herein.

Enzymes of the allulose production pathways as provided herein aretypically heterologous to the host cell (initially cloned from orobtained from a different cell type), although some of the enzymes maybe endogenous (native) to the host cell. Thus, in some embodiments, atleast one enzyme (e.g., thermostable enzyme) used to convert starchand/or cellodextrin to allulose is heterologous to the host cell. Insome embodiments, at least two, at least three, or at least four enzymesare heterologous to the host cell. In some embodiments, at least oneenzyme is endogenous (native) to the host cell. In some embodiments, atleast two, at least three, or at least four enzymes are endogenous tothe host cell.

The host cells may be prokaryotic cells, such as bacterial cells (e.g.,Escherichia coli cells), or eukaryotic cells, such as yeast cells orplant cells. Other cell types are described below.

In some embodiments, at least one of the enzymes used to convert starchand/or cellulose/cellodextrin to allulose is a thermostable enzyme. Insome embodiments, at least two (e.g., at least three or at least four)of the enzymes are thermostable enzymes. In some embodiments, all of theenzymes are thermostable enzymes. Thus, in some embodiments, the methodsinclude culturing cells engineered to express at least one thermostableα-glucan phosphorylase, at least one thermostable phosphoglucomutase, atleast one thermostable phosphoglucoisomerase, at least one thermostableallulose 6-phosphate epimerase, at least one thermostable allulose6-phosphate phosphatase, or a combination of at least two or more of theforegoing thermostable enzymes. In some embodiments, the methods includeculturing cells engineered to express at least one thermostablecellodextrin phosphorylase, at least one thermostablephosphoglucomutase, at least one thermostable phosphoglucoisomerase, atleast one thermostable allulose 6-phosphate epimerase, at least onethermostable allulose 6-phosphate phosphatase, or a combination of atleast two or more of the foregoing thermostable enzymes.

In some embodiments, the methods of producing allulose include lysing(e.g., thermal, osmotic, mechanical, chemical, or enzymatic lysis) thecultured cells to produce at least one (e.g., at least two, at leastthree, or at least four) cell lysate. It should be understood thatmultiple cell lysates (and thus multiple cell populations, e.g., fromthe same organism (e.g., bacteria) or from different organisms (e.g.,bacteria, yeast, and/or plant cells)) may be used in an enzymaticreaction as provided herein. For example, one cell population may beengineered to express one or more enzymes(s) of the allulose productionpathway, while another cell population (or several other cellpopulations) may be engineered to express another (at least one other)enzyme of the allulose production pathway. Thus, in some embodiments,the methods comprise culturing at least one population of cellsengineered to express at least one α-glucan phosphorylase, culturing atleast one cell population engineered to express at least onephosphoglucomutase, culturing at least one cell population engineered toexpress at least one phosphoglucoisomerase, culturing at least one cellpopulation engineered to express at least one allulose 6-phosphateepimerase, and/or culturing at least one cell population engineered toexpress at least one allulose 6-phosphate phosphatase. In someembodiments, the methods comprise culturing at least one population ofcells engineered to express at least one cellodextrin phosphorylase,culturing at least one cell population engineered to express at leastone phosphoglucomutase, culturing at least one cell populationengineered to express at least one phosphoglucoisomerase, culturing atleast one cell population engineered to express at least one allulose6-phosphate epimerase, and/or culturing at least one cell populationengineered to express at least one allulose 6-phosphate phosphatase.Following lysis of the cells, the cell lysates are combined such thatthe enzymes are present in a single cell lysate/reaction mixture.

It should be understood that in any one of the methods described herein,the cells may be lysed by any means, including mechanical, chemical,enzymatic, osmotic and/or thermal lysis. Thus, a lysing step and aheating (heat inactivation) step may be combined as a single step ofheating the cells to a temperature that lyses the cells and inactivatesundesired native enzymatic activities.

In some embodiments, the methods further include heating the celllysate(s) (or a cell lysate mixture) to a temperature that inactivatesundesired native enzymatic activities but does not inactivate any of thethermostable enzymes of the production pathway, to produce aheat-inactivated lysate. The cell lysate(s), in some embodiments, isheated to a temperature of at least 50° C. For example, the celllysate(s) may be heated to a temperature of at least 55° C., 60° C., 65°C., 70° C., 75° C., 80° C., 85° C., or 90° C. A native enzyme (or othernon-thermostable enzyme) is considered inactive, in some embodiments,when its level of activity is reduced by at least 50%. In someembodiments, a native enzyme (or other non-thermostable enzyme) isconsidered inactive when its level of activity is reduced by at least55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%.

The cell lysate(s) may be heated for a period of time sufficient toinactive native enzymes (or other non-thermostable enzymes) of the cell.For example, the cell lysate(s) may be heated for at least 2, 3, 4, or 5minutes. In some embodiments, the cell lysate(s) are heated for longerthan 5 minutes. In some embodiments, the cell lysate(s) are heated for aperiod of time sufficient to reduce the activity of at least some of thenative enzymes (or other non-thermostable enzymes) by at least 50%(e.g., at least 60%, 70%, 80%, or 90%).

Following heat inactivation, in some embodiments, at least one (e.g., atleast two or at least three) purified enzyme (or partially purifiedenzyme) is added to the cell lysate/reaction mixture. Thus, a reactionmixture, in some embodiments, includes a combination of enzymes presentin the cell lysate (expressed by the engineered host cell(s)) and atleast one purified enzyme. At least one purified enzyme may be selectedfrom the group consisting of α-glucan phosphorylases or cellodextrinphosphorylases, phosphoglucomutases, phosphoglucoisomerases, allulose6-phosphate epimerases, and allulose 6-phosphate phosphatases.

In some embodiments, the methods also include incubating theheat-inactivated lysate(s) in the presence of a starch and inorganicphosphate to produce allulose. In some embodiments, the methods alsoinclude incubating the heat-inactivated lysate(s) in the presence of acellodextrin and inorganic phosphate to produce allulose. In someembodiments, the heat-inactivated lysates are incubated at a temperatureof at least 50 C. In some embodiments, the heat-inactivated lysates areincubated for at least 2 minutes (e.g., at least 3, 4, or 5 minutes).For example, the heat-inactivated lysates may be incubated for 2-5minutes, or 2-10 minutes. The starch may be, for example, amylose,amylopectin, or a mixture of amylose and amylopectin. In someembodiments, biomass is used instead of starch. For example, thereaction may include cellodextrin phosphorylase(s). In some embodiments,the starch or cellodextrin is present as a component of a compound(e.g., part of the biomass). For example, in some embodiments, theheat-inactivated lysate(s) (e.g., microbial cell lysates) are incubatedin the presence of corn pulp and inorganic phosphate to produce allulose(or any other sugar described herein).

Also provided herein are cells and cell lysates used for the productionof allulose. Thus, an engineered cell (e.g., bacterial cell, yeast cell,and/or plant cell) or cell lysate(s) of the present disclosure mayinclude at least one (e.g., at least two, at least three, or at leastfour) enzyme selected from the group consisting of α-glucanphosphorylases, phosphoglucomutases, phosphoglucoisomerases, allulose6-phosphate epimerases, and allulose 6-phosphate phosphatases. In someembodiments, an engineered cell (e.g., bacterial cell and/or yeast cell)or cell lysate(s) of the present disclosure may include at least one(e.g., at least two, at least three, or at least four) enzyme selectedfrom the group consisting of cellodextrin phosphorylases,phosphoglucomutases, phosphoglucoisomerases, allulose 6-phosphateepimerases, and allulose 6-phosphate phosphatases. In some embodiments,an engineered cell (e.g., bacterial cell, yeast cell, and/or plant cell)or cell lysate(s) of the present disclosure includes at least one (e.g.,at least two, at least three, or at least four) enzyme selected from thegroup consisting of thermostable α-glucan phosphorylases, thermostablephosphoglucomutases, thermostable phosphoglucoisomerases, thermostableallulose 6-phosphate epimerases, and thermostable allulose 6-phosphatephosphatases. In some embodiments, an engineered cell (e.g., bacterialcell and/or yeast cell) or cell lysate(s) of the present disclosureincludes at least one (e.g., at least two, at least three, or at leastfour) enzyme selected from the group consisting of thermostablecellodextrin phosphorylases, thermostable phosphoglucomutases,thermostable phosphoglucoisomerases, thermostable allulose 6-phosphateepimerases, and thermostable allulose 6-phosphate phosphatases.

TABLE 2 Exemplary Allulose Pathway Enzymes Pathway Step Enzyme Name ECNo. Native Organism NCBI No. 1 α-glucan 2.4.1.1 Aquifex aeolicusWP_010880430 phosphorylase Thermocrinis minervae WP_079654205Thermosulfidibacter takaii WP_068550435 Thermosulfurimonas WP_068671361dismutans Thermococcus litoralis WP_004066514 Palaeococcus pacificusWP_048164525 Thermotoga neapolitana WP_015919877 RuminiclostridiumWP_003512623 thermocellum Pyrococcus abyssi WP_048146597 Thermococcusthioreducens WP_055429034 Deinococcus radiodurans NP_295917 Sulfolobusacidocaldarius WP_011277212 Thermus caldophilus AAV68178 Meiothermussilvanus ADH63988 Oceanithermus profundus WP_013457575 Ardenticatenamaritima WP_054491617 Thermococcus barophilus WP_013466486Pseudothermotoga WP_013931760 thermarum Hydrogenobacter WP_012962737thermophilus Thermus oshimai AFV76231 Meiothermus ruber WP_013013285Marinitoga piezophila WP_014295659 Cellodextrin 2.4.2.49 Clostridiumthermocellum BAB71818 phosphorylase (also referred to as cellodextrinphosphorylase) Clostridium straminisolvens GAE90338 Thermotoga RQ2WP_011943512 Ignisphaera aggregans ADM28607 Thermotoga maritimaWP_004082399 Spirochaeta thermophila WP_013314871 Caldicellulosiruptorbescii WP_015907054 Dictyoglomus thermophilum WP_012548338Thermoanaerobacterium WP_013297089 thermosaccharolyticum Thermosiphoafricanus ACJ76363 Caldisalinibacter WP_006313788 kiritimatiensisDefluviitalea phaphyphila WP_058486419 Caldicellulosiruptor WP_013429146kronotskyensis Thermococcus sibiricus WP_015848606 Thermosphaeraaggregans WP_013129904 2 Phosphoglucomutase 5.4.2.6 ThermococcusBAD42440 kodakaraensis Pyrococcus kukulkanii WP_068320630 Ammonifexdegensii WP_015738524 Methanothermobacter WP_074359679 wolfeiiMethanothermus fervidus WP_013413625 Sulfolobus acidocaldariusWP_011277678 Archaeoglobus fulgidus WP_010877965 Ferroglobus placidusWP_012964640 Geoglobus ahangari WP_048096365 Archaeoglobus veneficusWP_013683858 Archaeoglobus WP_015589873 sulfaticallidus Aciduliprofundumboonie WP_012997480 Clostridium thermocellum WP_003517493 Defluviitaleaphaphyphila WP_058485855 Caminicella sporogenes WP_072968430Caloranaerobacter WP_069650396 ferrireducens Thermosipho malanesiensisWP_012056981 Fervidobacterium WP_014451812 pennivorans SymbiobacteriumWP_011196853 thermophilum Spirochaeta thermophila ADN02136Thermoanaerobacter AEM79998 wiegelii 3 Phosphoglucoisomerase 5.3.1.9Thermus thermophilus WP_041443619 Meiothermus timidus WP_018467230Thermus filiformis WP_038061840 Marinithermus WP_013704730hydrothermalis Thermosipho africanus WP_004103575 SulfurihydrogenibiumWP_012674892 azorense Persephonella marina WP_012675923 Marinitogapiezophila WP_014295589 Kosmotoga olearia WP_012744692 Thermotogamaritima WP_004081585 Geobacillus KZE97846 stearothermophilusAnoxybacillus flavithermus WP_041638934 Thermosulfidibacter takaiiBAT72177 Fervidobacterium nodosum WP_011994042 Clostridium thermocellumWP_003512317 Thermoanaerobacterium WP_013297353 thermosaccharolyticumMethanococcus jannaschii WP_010871130 Methanotorris igneus WP_013799854Methanocaldococcus villosus WP_004589908 MethanothermococcusWP_013867340 okinawensis Pseudothermotoga WP_013931655 thermarumDeferribacter desulfuricans WP_013007743 Thermovibrio ammonificansWP_013537727 4 Allulose 6- 5.3.1.— Thermobacterium WP_013298194phosphate thermosaccharolyticum epimerase Thermoanaerobacter brockiiWP_003868217 Caldanaerobacter WP_011025758 subterraneus Deferribacterdesulfuricans WP_013008817 Thermocrinis ruber WP_025305325Hydrogenivirga sp. 128-5- WP_008287078 R1-1 Brevibacillus thermoruberWP_029098887 Thermosipho atlanticus WP_073071389 Thermosulfidibactertakaii WP_068550718 5 Allulose 6- 3.1.3.— ThermoanaerobacterWP_014063120 phosphate wiegelii phosphatase ThermoanaerobacterWP_003870772 ethanolicus Thermus islandicus WP_022799086 Deinococcusgeothermalis ABF44399 DSM 11300 Thermosphaera aggregans WP_013129214Crenarchaeota archaeon WP_013335457 13_1_40CM_3_53_5 Pyrococcushorikoshii Ot3 WP_010884566 Aquifex aeolicus WP_010880861Ruminiclostridium WP_003512401 thermocellum Desulfotomaculum AEG14852kuznetsovii Caldanaerobacter WP_009610632 subterraneus Acidothermuscellulolyticus WP_011718939 Methanothermobacter WP_010877362thermautotrophicus Thermobifida fusca AAZ54262 Thermotoga neapolitanaACM23496 Petrotoga mobilis WP_012207996 Thermodesulfatator indicusWP_013908370 Thermus thermophilus AAS81813 Bacteroides vulgatus ABR41712Bacteroides fragilis CAH06673

It should be understood that the pathway for producing allulose may beinclude any combination of enzymes selected from each of Pathways Steps1-5 of Table 2. For example, the α-glucan phosphorylase of Pathway Step1 may be selected from any one of the α-glucan phosphorylases of Aquifexaeolicus, Thermocrinis minervae, Thermosulfidibacter takaii,Thermosulfurimonas dismutans, Thermococcus litoralis, Palaeococcuspacificus, Thermotoga neapolitana, Ruminiclostridium thermocellum,Pyrococcus abyssi, Thermococcus thioreducens, Deinococcus radiodurans,Sulfolobus acidocaldarius, Thermus caldophilus, Meiothermus silvanus,Oceanithermus profundus, Ardenticatena maritima, Thermococcusbarophilus, Pseudothermotoga thermarum, Hydrogenobacter thermophilus,Thermus oshimai, Meiothermus ruber, and Marinitoga piezophila andcombined with a phosphoglucomutase of Pathway Step 2 selected from anyone of the phosphoglucomutase of Thermococcus kodakaraensis, Pyrococcuskukulkanii, Ammonifex degensii, Methanothermobacter wolfeii,Methanothermus fervidus, Sulfolobus acidocaldarius, Archaeoglobusfulgidus, Ferroglobus placidus, Geoglobus ahangari, Archaeoglobusveneficus, Archaeoglobus sulfaticallidus, Aciduliprofundum boonie,Clostridium thermocellum, Defluviitalea phaphyphila, Caminicellasporogenes, Caloranaerobacter ferrireducens, Thermosipho malanesiensis,Fervidobacterium pennivorans, Symbiobacterium thermophilum, Spirochaetathermophila, and Thermoanaerobacter wiegelii.

Glucose Production

Other aspects of the present disclosure provide methods, compositions,and systems for producing glucose. These methods, in some embodiments,include culturing cells engineered to express at least one α-glucanphosphorylase and/or at least one cellodextrin phosphorylase, at leastone phosphoglucomutase, at least one glucose 6-phosphate phosphatase, ora combination of at least two of the foregoing enzymes. In someembodiments, the α-glucan phosphorylase (and/or cellodextrinphosphorylase) and the phosphoglucomutase are expressed as a singlefusion (chimeric) protein or a bifunctional protein. In someembodiments, these methods include culturing cells engineered to expressat least one α-glucan phosphorylase and/or at least one cellodextrinphosphorylase, at least one glucose 1-phosphate phosphatase, or acombination of at least two of the foregoing enzymes. In someembodiments, the α-glucan phosphorylase (and/or cellodextrinphosphorylase) and the phosphoglucomutase are expressed as a singlefusion (chimeric) protein or a bifunctional protein.

Enzymes of the glucose production pathways as provided herein aretypically heterologous to the host cell (initially cloned from orobtained from a different cell type), although some of the enzymes maybe endogenous (native) to the host cell. Thus, in some embodiments, atleast one enzyme (e.g., thermostable enzyme) used to convert starchand/or cellodextrin to glucose is heterologous to the host cell. In someembodiments, at least two enzymes are heterologous to the host cell. Insome embodiments, at least one enzyme (e.g., thermostable enzyme) isendogenous (native) to the host cell. In some embodiments, at least twoenzymes are endogenous to the host cell.

The host cells may be prokaryotic cells, such as bacterial cells (e.g.,Escherichia coli cells), or eukaryotic cells, such as yeast cells orplant cells. Other cell types are described below.

In some embodiments, at least one of the enzymes used to convert starchand/or cellodextrin to glucose is a thermostable enzyme. In someembodiments, at least two of the enzymes are thermostable enzymes. Insome embodiments, all of the enzymes are thermostable enzymes. Thus, insome embodiments, the methods include culturing cells engineered toexpress at least one thermostable α-glucan phosphorylase, at least onethermostable phosphoglucomutase, at least one thermostable glucose6-phosphate phosphatase, or a combination of at least two or more of theforegoing thermostable enzymes. In some embodiments, the methods includeculturing cells engineered to express at least one thermostablecellodextrin phosphorylase, at least one thermostablephosphoglucomutase, at least one thermostable glucose 6-phosphatephosphatase, or a combination of at least two or more of the foregoingthermostable enzymes. In some embodiments, the methods include culturingcells engineered to express at least one thermostable α-glucanphosphorylase, at least one thermostable glucose 1-phosphatephosphatase, or a combination of at least two or more of the foregoingthermostable enzymes. In some embodiments, the methods include culturingcells engineered to express at least one thermostable cellodextrinphosphorylase, at least one thermostable glucose 1-phosphatephosphatase, or a combination of at least two or more of the foregoingthermostable enzymes.

In some embodiments, the methods of producing glucose include lysing(e.g., thermal, mechanical, chemical, or enzymatic lysis) the culturedcells to produce at least one (e.g., at least two, at least three, or atleast four) cell lysate. It should be understood that multiple celllysates (and thus multiple cell populations, e.g., from the sameorganism (e.g., bacteria) or from different organisms (e.g., bacteria,yeast and or plant cells)) may be used in an enzymatic reaction asprovided herein. For example, one cell population may be engineered toexpress one or more enzymes(s) of the glucose production pathway, whileanother cell population (or several other cell populations) may beengineered to express another (at least one other) enzyme of the glucoseproduction pathway. Thus, in some embodiments, the methods compriseculturing at least one population of cells engineered to express atleast one α-glucan phosphorylase, culturing at least one cell populationengineered to express at least one phosphoglucomutase, and/or culturingat least one cell population engineered to express at least one glucose6-phosphate phosphatase. In some embodiments, the methods compriseculturing at least one population of cells engineered to express atleast one cellodextrin phosphorylase, culturing at least one cellpopulation engineered to express at least one phosphoglucomutase, and/orculturing at least one cell population engineered to express at leastone glucose 6-phosphate phosphatase. In some embodiments, the methodscomprise culturing at least one population of cells engineered toexpress at least one α-glucan phosphorylase, and/or culturing at leastone cell population engineered to express at least one glucose1-phosphate phosphatase. In some embodiments, the methods compriseculturing at least one population of cells engineered to express atleast one cellodextrin phosphorylase, and/or culturing at least one cellpopulation engineered to express at least one glucose 1-phosphatephosphatase. Following lysis of the cells, the cell lysates are combinedsuch that the enzymes are present in a single cell lysate/reactionmixture.

In some embodiments, the methods further include heating the celllysate(s) (or a cell lysate mixture) to a temperature that inactivatesundesired native enzymatic activities but does not inactivate any of thethermostable enzymes of the production pathway, to produce aheat-inactivated lysate. The cell lysate(s), in some embodiments, isheated to a temperature of at least 50° C. For example, the celllysate(s) may be heated to a temperature of at least 55° C., 60° C., 65°C., 70° C., 75° C., 80° C., 85° C., or 90° C.

The cell lysate(s) may be heated for a period of time sufficient toinactive native enzymes (or other non-thermostable enzymes) of the cell.For example, the cell lysate(s) may be heated for at least 2, 3, 4, orat least 5 minutes. In some embodiments, the cell lysate(s) are heatedfor longer than 5 minutes. In some embodiments, the cell lysate(s) areheated for a period of time sufficient to reduce the activity of nativeenzymes (or other non-thermostable enzymes) by at least 50% (e.g., atleast 60%, 70%, 80%, or 90%).

Following heat inactivation, in some embodiments, at least one (e.g., atleast two or at least three) purified enzymes is added to the celllysate/reaction mixture. Thus, a reaction mixture, in some embodiments,includes a combination of enzymes present in the cell lysate (expressedby the engineered host cell(s)) and at least one purified enzyme. Atleast one purified enzyme may be selected from the group consisting ofα-glucan phosphorylases or cellodextrin phosphorylases,phosphoglucomutases, and glucose 6-phosphate phosphatases.Alternatively, at least one purified enzyme may be selected from thegroup consisting of α-glucan phosphorylases or cellodextrinphosphorylases and glucose 1-phosphate phosphatases.

In some embodiments, the methods also include incubating theheat-inactivated lysate(s) in the presence of a starch and inorganicphosphate to produce glucose. In some embodiments, the methods alsoinclude incubating the heat-inactivated lysate(s) in the presence of acellodextrin and inorganic phosphate to produce glucose. In someembodiments, the heat-inactivated lysates are incubated at a temperatureof at least 50 C. In some embodiments, the heat-inactivated lysates areincubated for at least 2 minutes (e.g., at least 3, 4, or 5 minutes).For example, the heat-inactivated lysates may be incubated for 2-5minutes, or 2-10 minutes.

The starch may be, for example, amylose, amylopectin, or a mixture ofamylose and amylopectin. In some embodiments, biomass is used instead ofstarch. In these embodiments, the reaction includes cellodextrinphosphorylase(s). In some embodiments, the starch or cellodextrin ispresent as a component of a compound.

Also provided herein are cells and cell lysates used for the productionof glucose. Thus, an engineered cell (e.g., bacterial cell and/or yeastcell) or cell lysate(s) of the present disclosure may include at leastone (e.g., at least two) enzyme selected from the group consisting ofα-glucan phosphorylases, phosphoglucomutases, and glucose 6-phosphatephosphatases. In some embodiments, an engineered cell (e.g., bacterialcell and/or yeast cell) or cell lysate(s) of the present disclosure mayinclude at least one (e.g., at least two) enzyme selected from the groupconsisting of cellodextrin phosphorylases, phosphoglucomutases, andglucose 6-phosphate phosphatases. An engineered cell (e.g., bacterialcell and/or yeast cell) or cell lysate(s) of the present disclosure mayinclude at least one (e.g., at least two) enzyme selected from the groupconsisting of α-glucan phosphorylases and glucose 1-phosphatephosphatases. In some embodiments, an engineered cell (e.g., bacterialcell and/or yeast cell) or cell lysate(s) of the present disclosure mayinclude at least one (e.g., at least two) enzyme selected from the groupconsisting of cellodextrin phosphorylases and glucose 1-phosphatephosphatases. In some embodiments, an engineered cell (e.g., bacterialcell and/or yeast cell) or cell lysate(s) of the present disclosureincludes at least one (e.g., at least two) enzyme selected from thegroup consisting of thermostable α-glucan phosphorylases, thermostablephosphoglucomutases, and thermostable glucose 6-phosphate phosphatases.In some embodiments, an engineered cell (e.g., bacterial cell and/oryeast cell) or cell lysate(s) of the present disclosure includes atleast one (e.g., at least two) enzyme selected from the group consistingof thermostable cellodextrin phosphorylases, thermostablephosphoglucomutases, and thermostable glucose 6-phosphate phosphatases.In some embodiments, an engineered cell (e.g., bacterial cell and/oryeast cell) or cell lysate(s) of the present disclosure includes atleast one (e.g., at least two) enzyme selected from the group consistingof thermostable α-glucan phosphorylases and thermostable glucose1-phosphate phosphatases. In some embodiments, an engineered cell (e.g.,bacterial cell and/or yeast cell) or cell lysate(s) of the presentdisclosure includes at least one (e.g., at least two) enzyme selectedfrom the group consisting of thermostable cellodextrin phosphorylasesand thermostable glucose 1-phosphate phosphatases.

Fructose Production

Yet other aspects of the present disclosure provide methods,compositions, and systems for producing fructose. These methods, in someembodiments, include culturing cells engineered to express at least oneα-glucan phosphorylase and/or cellodextrin phosphorylase, at least onephosphoglucomutase, at least one phosphoglucoisomerase, at least onefructose 6-phosphate phosphatase, or a combination of at least two ofthe foregoing enzymes. In some embodiments, the α-glucan phosphorylase(and/or cellodextrin phosphorylase) and the phosphoglucomutase areexpressed as a single fusion (chimeric) protein or a bifunctionalprotein.

Enzymes of the fructose production pathways as provided herein aretypically heterologous to the host cell (initially cloned from orobtained from a different cell type), although some of the enzymes maybe endogenous (native) to the host cell. Thus, in some embodiments, atleast one enzyme (e.g., thermostable enzyme) used to convert starchand/or cellodextrin to fructose is heterologous to the host cell. Insome embodiments, at least two or at least three enzymes areheterologous to the host cell. In some embodiments, at least one enzyme(e.g., thermostable enzyme) is endogenous (native) to the host cell. Insome embodiments, at least two or at least three enzymes are endogenousto the host cell.

The host cells may be prokaryotic cells, such as bacterial cells (e.g.,Escherichia coli cells), or eukaryotic cells, such as yeast cells orplant cells. Other cell types are described below.

In some embodiments, at least one of the enzymes used to convert starchand/or cellodextrin to fructose is a thermostable enzyme. In someembodiments, at least two or at least three of the enzymes arethermostable enzymes. In some embodiments, all of the enzymes arethermostable enzymes. Thus, in some embodiments, the methods includeculturing cells engineered to express at least one thermostable α-glucanphosphorylase, at least one thermostable phosphoglucomutase, at leastone thermostable phosphoglucoisomerase, at least one thermostablefructose 6-phosphate phosphatase, or a combination of at least two ormore of the foregoing thermostable enzymes. In some embodiments, themethods include culturing cells engineered to express at least onethermostable cellodextrin phosphorylase, at least one thermostablephosphoglucomutase, at least one thermostable phosphoglucoisomerase, atleast one thermostable fructose 6-phosphate phosphatase, or acombination of at least two or more of the foregoing thermostableenzymes.

In some embodiments, the methods of producing fructose include lysing(e.g., thermal, mechanical, chemical, or enzymatic lysis) the culturedcells to produce at least one (e.g., at least two, at least three, or atleast four) cell lysate. It should be understood that multiple celllysates (and thus multiple cell populations, e.g., from the sameorganism (e.g., bacteria) or from different organisms (e.g., bacteria,yeast and or plant cells)) may be used in an enzymatic reaction asprovided herein. For example, one cell population may be engineered toexpress one or more enzymes(s) of the fructose production pathway, whileanother cell population (or several other cell populations) may beengineered to express another (at least one other) enzyme of thefructose production pathway. Thus, in some embodiments, the methodscomprise culturing at least one population of cells engineered toexpress at least one α-glucan phosphorylase, culturing at least one cellpopulation engineered to express at least one phosphoglucomutase,culturing at least one cell population engineered to express at leastone phosphoglucoisomerase, and/or culturing at least one cell populationengineered to express at least one fructose 6-phosphate phosphatase. Insome embodiments, the methods comprise culturing at least one populationof cells engineered to express at least one cellodextrin phosphorylase,culturing at least one cell population engineered to express at leastone phosphoglucomutase, culturing at least one cell populationengineered to express at least one phosphoglucoisomerase, and/orculturing at least one cell population engineered to express at leastone fructose 6-phosphate phosphatase. Following lysis of the cells, thecell lysates are combined such that the enzymes are present in a singlecell lysate/reaction mixture.

In some embodiments, the methods further include heating the celllysate(s) (or a cell lysate mixture) to a temperature that inactivatesundesired native enzymatic activities but does not inactivate any of thethermostable enzymes of the production pathway, to produce aheat-inactivated lysate. The cell lysate(s), in some embodiments, isheated to a temperature of at least 50° C. For example, the celllysate(s) may be heated to a temperature of at least 55° C., 60° C., 65°C., 70° C., 75° C., 80° C., 85° C., or 90° C.

The cell lysate(s) may be heated for a period of time sufficient toinactive native enzymes (or other non-thermostable enzymes) of the cell.For example, the cell lysate(s) may be heated for at least 2, 3, 4, orat least 5 minutes. In some embodiments, the cell lysate(s) are heatedfor longer than 5 minutes. In some embodiments, the cell lysate(s) areheated for a period of time sufficient to reduce the activity of nativeenzymes (or other non-thermostable enzymes) by at least 50% (e.g., atleast 60%, 70%, 80%, or 90%).

Following heat inactivation, in some embodiments, at least one (e.g., atleast two or at least three) purified enzymes is added to the celllysate/reaction mixture. Thus, a reaction mixture, in some embodiments,includes a combination of enzymes present in the cell lysate (expressedby the engineered host cell(s)) and at least one purified enzyme. Atleast one purified enzyme may be selected from the group consisting ofα-glucan phosphorylases or cellodextrin phosphorylases,phosphoglucomutases, phosphoglucoisomerases, and fructose 6-phosphatephosphatases.

In some embodiments, the methods also include incubating theheat-inactivated lysate(s) in the presence of a starch and inorganicphosphate to produce fructose. In some embodiments, the methods alsoinclude incubating the heat-inactivated lysate(s) in the presence of acellodextrin and inorganic phosphate to produce fructose. In someembodiments, the heat-inactivated lysates are incubated at a temperatureof at least 50 C. In some embodiments, the heat-inactivated lysates areincubated for at least 2 minutes (e.g., at least 3, 4, or 5 minutes).For example, the heat-inactivated lysates may be incubated for 2-5minutes, or 2-10 minutes. The starch may be, for example, amylose,amylopectin, or a mixture of amylose and amylopectin. In someembodiments, biomass is used instead of starch. In these embodiments,the reaction includes cellodextrin phosphorylase(s). In someembodiments, the starch or cellodextrin is present as a component of acompound.

Also provided herein are cells and cell lysates used for the productionof fructose. Thus, an engineered cell (e.g., bacterial cell and/or yeastcell) or cell lysate(s) of the present disclosure may include at leastone (e.g., at least two or at least three) enzyme selected from thegroup consisting of α-glucan phosphorylases, phosphoglucomutases, andfructose 6-phosphate phosphatases. In some embodiments, an engineeredcell (e.g., bacterial cell and/or yeast cell) or cell lysate(s) of thepresent disclosure includes at least one (e.g., at least two or at leastthree) enzyme selected from the group consisting of thermostableα-glucan phosphorylases, thermostable phosphoglucomutases, andthermostable fructose 6-phosphate phosphatase. In some embodiments, anengineered cell (e.g., bacterial cell and/or yeast cell) or celllysate(s) of the present disclosure includes at least one (e.g., atleast two or at least three) enzyme selected from the group consistingof thermostable cellodextrin phosphorylases, thermostablephosphoglucomutases, and thermostable fructose 6-phosphate phosphatase.

Mannose Production

Further still, some aspects of the present disclosure provide methods,compositions, and systems for producing mannose. These methods, in someembodiments, include culturing cells engineered to express at least oneα-glucan phosphorylase and/or at least one cellodextrin phosphorylase,at least one phosphoglucomutase, at least one phosphoglucoisomerase, atleast one mannose 6-phosphate isomerase, at least one mannose6-phosphate phosphatase, or a combination of at least two of theforegoing enzymes. In some embodiments, the α-glucan phosphorylase(and/or cellodextrin phosphorylase) and the phosphoglucomutase areexpressed as a single fusion (chimeric) protein or a bifunctionalprotein.

Enzymes of the mannose production pathways as provided herein aretypically heterologous to the host cell (initially cloned from orobtained from a different cell type), although some of the enzymes maybe endogenous (native) to the host cell. Thus, in some embodiments, atleast one enzyme (e.g., thermostable enzyme) used to convert starchand/or cellodextrin to mannose is heterologous to the host cell. In someembodiments, at least two, at least three, at least four, at least five,or at least six enzymes are heterologous to the host cell. In someembodiments, at least one enzyme (e.g., thermostable enzyme) isendogenous (native) to the host cell. In some embodiments, at least two,at least three, at least four, at least five, or at least six enzymesare endogenous to the host cell.

The host cells may be prokaryotic cells, such as bacterial cells (e.g.,Escherichia coli cells), or eukaryotic cells, such as yeast cells orplant cells. Other cell types are described below.

In some embodiments, at least one of the enzymes used to convert starchand/or cellodextrin to mannose is a thermostable enzyme. In someembodiments, at least two, at least three, at least four, at least five,or at least six of the enzymes are thermostable enzymes. In someembodiments, all of the enzymes are thermostable enzymes. Thus, in someembodiments, the methods include culturing cells engineered to expressat least one thermostable α-glucan phosphorylase, at least onethermostable phosphoglucomutase, at least one thermostablephosphoglucoisomerase, at least one thermostable mannose 6-phosphateisomerase, at least one thermostable mannose 6-phosphate phosphatase, ora combination of at least two or more of the foregoing thermostableenzymes. In some embodiments, the methods include culturing cellsengineered to express at least one thermostable cellodextrinphosphorylase, at least one thermostable phosphoglucomutase, at leastone thermostable phosphoglucoisomerase, at least one thermostablemannose 6-phosphate isomerase, at least one thermostable mannose6-phosphate phosphatase, or a combination of at least two or more of theforegoing thermostable enzymes.

In some embodiments, the methods of producing mannose include lysing(e.g., thermal, mechanical, chemical, or enzymatic lysis) the culturedcells to produce at least one (e.g., at least two, at least three, or atleast four) cell lysate. It should be understood that multiple celllysates (and thus multiple cell populations, e.g., from the sameorganism (e.g., bacteria) or from different organisms (e.g., bacteria,yeast and or plant cells)) may be used in an enzymatic reaction asprovided herein. For example, one cell population may be engineered toexpress one or more enzymes(s) of the mannose production pathway, whileanother cell population (or several other cell populations) may beengineered to express another (at least one other) enzyme of the mannoseproduction pathway. Thus, in some embodiments, the methods compriseculturing at least one population of cells engineered to express atleast one α-glucan phosphorylase, culturing at least one cell populationengineered to express at least one phosphoglucomutase, culturing atleast one cell population engineered to express at least onephosphoglucoisomerase, culturing at least one cell population engineeredto express at least one mannose 6-phosphate isomerase, and/or culturingat least one cell population engineered to express at least one mannose6-phosphate phosphatase. In some embodiments, the methods compriseculturing at least one population of cells engineered to express atleast one cellodextrin phosphorylase, culturing at least one cellpopulation engineered to express at least one phosphoglucomutase,culturing at least one cell population engineered to express at leastone phosphoglucoisomerase, culturing at least one cell populationengineered to express at least one mannose 6-phosphate isomerase, and/orculturing at least one cell population engineered to express at leastone mannose 6-phosphate phosphatase. Following lysis of the cells, thecell lysates are combined such that the enzymes are present in a singlecell lysate/reaction mixture.

In some embodiments, the methods further include heating the celllysate(s) (or a cell lysate mixture) to a temperature that inactivatesundesired native enzymatic activities but does not inactivate any of thethermostable enzymes of the production pathway, to produce aheat-inactivated lysate. The cell lysate(s), in some embodiments, isheated to a temperature of at least 50° C. For example, the celllysate(s) may be heated to a temperature of at least 55° C., 60° C., 65°C., 70° C., 75° C., 80° C., 85° C., or 90° C.

The cell lysate(s) may be heated for a period of time sufficient toinactive native enzymes (or other non-thermostable enzymes) of the cell.For example, the cell lysate(s) may be heated for at least 2, 3, 4, orat least 5 minutes. In some embodiments, the cell lysate(s) are heatedfor longer than 5 minutes. In some embodiments, the cell lysate(s) areheated for a period of time sufficient to reduce the activity of nativeenzymes (or other non-thermostable enzymes) by at least 50% (e.g., atleast 60%, 70%, 80%, or 90%).

Following heat inactivation, in some embodiments, at least one (e.g., atleast two or at least three) purified enzymes is added to the celllysate/reaction mixture. Thus, a reaction mixture, in some embodiments,includes a combination of enzymes present in the cell lysate (expressedby the engineered host cell(s)) and at least one purified enzyme. Atleast one purified enzyme may be selected from the group consisting ofα-glucan phosphorylases or cellodextrin phosphorylases,phosphoglucomutases, phosphoglucoisomerase, mannose 6-phosphateisomerases, and mannose 6-phosphate phosphatases.

In some embodiments, the methods also include incubating theheat-inactivated lysate(s) in the presence of a starch and inorganicphosphate to produce mannose. In some embodiments, the methods alsoinclude incubating the heat-inactivated lysate(s) in the presence of acellodextrin and inorganic phosphate to produce mannose. In someembodiments, the heat-inactivated lysates are incubated at a temperatureof at least 50 C. In some embodiments, the heat-inactivated lysates areincubated for at least 2 minutes (e.g., at least 3, 4, or 5 minutes).For example, the heat-inactivated lysates may be incubated for 2-5minutes, or 2-10 minutes. The starch may be, for example, amylose,amylopectin, or a mixture of amylose and amylopectin. In someembodiments, biomass is used instead of starch. In these embodiments,the reaction includes cellodextrin phosphorylase(s). In someembodiments, the starch or cellodextrin is present as a component of acompound.

Also provided herein are cells and cell lysates used for the productionof mannose. Thus, an engineered cell (e.g., bacterial cell and/or yeastcell) or cell lysate(s) of the present disclosure may include at leastone (e.g., at least two, at least three, at least four, at least five,or at least six) enzyme selected from the group consisting of α-glucanphosphorylases, phosphoglucomutases, phosphoglucoisomerases, mannose6-phosphate isomerases, and mannose 6-phosphate phosphatases. Anengineered cell (e.g., bacterial cell and/or yeast cell) or celllysate(s) of the present disclosure may include at least one (e.g., atleast two, at least three, at least four, at least five, or at leastsix) enzyme selected from the group consisting of cellodextrinphosphorylases, phosphoglucomutases, phosphoglucoisomerases, mannose6-phosphate isomerases, and mannose 6-phosphate phosphatases. In someembodiments, an engineered cell (e.g., bacterial cell and/or yeast cell)or cell lysate(s) of the present disclosure includes at least one (e.g.,at least two, at least three, at least four, at least five, or at leastsix) enzyme selected from the group consisting of thermostable α-glucanphosphorylases, thermostable phosphoglucomutases, thermostablephosphoglucoisomerases, thermostable mannose 6-phosphate isomerases, andthermostable mannose 6-phosphate phosphatases. In some embodiments, anengineered cell (e.g., bacterial cell and/or yeast cell) or celllysate(s) of the present disclosure includes at least one (e.g., atleast two, at least three, at least four, at least five, or at leastsix) enzyme selected from the group consisting of thermostablecellodextrin phosphorylases, thermostable phosphoglucomutases,thermostable phosphoglucoisomerases, thermostable mannose 6-phosphateisomerases, and thermostable mannose 6-phosphate phosphatases.

Sorbitol Production

Still other aspects of the present disclosure provide methods,compositions, and systems for producing sorbitol. These methods, in someembodiments, include culturing cells engineered to express at least oneα-glucan phosphorylase and/or at least on cellodextrin phosphorylase, atleast one phosphoglucomutase, at least one aldose dehydrogenase, atleast one sorbitol-6-phosphate phosphatase, or a combination of at leasttwo of the foregoing enzymes. In some embodiments, the methods includeculturing cells engineered to express at least one α-glucanphosphorylase and/or at least on cellodextrin phosphorylase, at leastone phosphoglucomutase, at least one phosphoglucoisomerase, at least onesorbitol-6-phosphate 2-dehydrogenase, at least one sorbitol-6-phosphatephosphatase, or a combination of at least two of the foregoing enzymes.In some embodiments, the α-glucan phosphorylase (and/or cellodextrinphosphorylase) and the phosphoglucomutase are expressed as a singlefusion (chimeric) protein or a bifunctional protein.

Enzymes of the sorbitol production pathways as provided herein aretypically heterologous to the host cell (initially cloned from orobtained from a different cell type), although some of the enzymes maybe endogenous (native) to the host cell. Thus, in some embodiments, atleast one enzyme (e.g., thermostable enzyme) used to convert starchand/or cellodextrin to sorbitol is heterologous to the host cell. Insome embodiments, at least two or at least three enzymes areheterologous to the host cell. In some embodiments, at least one enzyme(e.g., thermostable enzyme) is endogenous (native) to the host cell. Insome embodiments, at least two or at least three enzymes are endogenousto the host cell.

The host cells may be prokaryotic cells, such as bacterial cells (e.g.,Escherichia coli cells), or eukaryotic cells, such as yeast cells orplant cells. Other cell types are described below.

In some embodiments, at least one of the enzymes used to convert starchand/or cellodextrin to sorbitol is a thermostable enzyme. In someembodiments, at least two, at least three, or at least four of theenzymes are thermostable enzymes. In some embodiments, all of theenzymes are thermostable enzymes. Thus, in some embodiments, the methodsinclude culturing cells engineered to express at least one thermostableα-glucan phosphorylase, at least one thermostable phosphoglucomutase, atleast one thermostable aldose dehydrogenase, at least one thermostablesorbitol-6-phosphate phosphatase, or a combination of at least two ormore of the foregoing thermostable enzymes. In some embodiments, themethods include culturing cells engineered to express at least onethermostable cellodextrin phosphorylase, at least one thermostablephosphoglucomutase, at least one thermostable aldose dehydrogenase, atleast one thermostable sorbitol-6-phosphate phosphatase, or acombination of at least two or more of the foregoing thermostableenzymes. In some embodiments, the methods include culturing cellsengineered to express at least one thermostable α-glucan phosphorylase,at least one thermostable phosphoglucomutase, at least one thermostablephosphoglucoisomerase, at least one thermostable sorbitol-6-phosphate2-dehydrogenase, at least one thermostable sorbitol-6-phosphatephosphatase, or a combination of at least two or more of the foregoingthermostable enzymes. In some embodiments, the methods include culturingcells engineered to express at least one thermostable cellodextrinphosphorylase, at least one thermostable phosphoglucomutase, at leastone thermostable phosphoglucoisomerase, at least one thermostablesorbitol-6-phosphate 2-dehydrogenase, at least one thermostablesorbitol-6-phosphate phosphatase, or a combination of at least two ormore of the foregoing thermostable enzymes.

In some embodiments, the methods of producing sorbitol include lysing(e.g., thermal, mechanical, chemical, or enzymatic lysis) the culturedcells to produce at least one (e.g., at least two, at least three, or atleast four) cell lysate. It should be understood that multiple celllysates (and thus multiple cell populations, e.g., from the sameorganism (e.g., bacteria) or from different organisms (e.g., bacteria,yeast and or plant cells)) may be used in an enzymatic reaction asprovided herein. For example, one cell population may be engineered toexpress one or more enzymes(s) of the sorbitol production pathway, whileanother cell population (or several other cell populations) may beengineered to express another (at least one other) enzyme of thesorbitol production pathway. Thus, in some embodiments, the methodscomprise culturing at least one population of cells engineered toexpress at least one α-glucan phosphorylase, culturing at least one cellpopulation engineered to express at least one phosphoglucomutase,culturing at least one cell population engineered to express at leastone aldose dehydrogenase, and/or culturing at least one cell populationengineered to express at least one sorbitol-6-phosphate phosphatase. Insome embodiments, the methods comprise culturing at least one populationof cells engineered to express at least one cellodextrin phosphorylase,culturing at least one cell population engineered to express at leastone phosphoglucomutase, culturing at least one cell populationengineered to express at least one aldose dehydrogenase, and/orculturing at least one cell population engineered to express at leastone sorbitol-6-phosphate phosphatase. Following lysis of the cells, thecell lysates are combined such that the enzymes are present in a singlecell lysate/reaction mixture.

In some embodiments, the methods further include heating the celllysate(s) (or a cell lysate mixture) to a temperature that inactivatesundesired native enzymatic activities but does not inactivate any of thethermostable enzymes of the production pathway, to produce aheat-inactivated lysate. The cell lysate(s), in some embodiments, isheated to a temperature of at least 50° C. For example, the celllysate(s) may be heated to a temperature of at least 55° C., 60° C., 65°C., 70° C., 75° C., 80° C., 85° C., or 90° C.

The cell lysate(s) may be heated for a period of time sufficient toinactive native enzymes (or other non-thermostable enzymes) of the cell.For example, the cell lysate(s) may be heated for at least 2, 3, 4, orat least 5 minutes. In some embodiments, the cell lysate(s) are heatedfor longer than 5 minutes. In some embodiments, the cell lysate(s) areheated for a period of time sufficient to reduce the activity of nativeenzymes (or other non-thermostable enzymes) by at least 50% (e.g., atleast 60%, 70%, 80%, or 90%).

Following heat inactivation, in some embodiments, at least one (e.g., atleast two or at least three) purified enzymes is added to the celllysate/reaction mixture. Thus, a reaction mixture, in some embodiments,includes a combination of enzymes present in the cell lysate (expressedby the engineered host cell(s)) and at least one purified enzyme. Atleast one purified enzyme may be selected from the group consisting ofα-glucan phosphorylases or cellodextrin phosphorylases,phosphoglucomutases, phosphoglucoisomerases, sorbitol-6-phosphate2-dehydrogenases, aldose dehydrogenases, and sorbitol-6-phosphatephosphatases.

In some embodiments, the methods also include incubating theheat-inactivated lysate(s) in the presence of a starch and inorganicphosphate to produce sorbitol. In some embodiments, the methods alsoinclude incubating the heat-inactivated lysate(s) in the presence of acellodextrin and inorganic phosphate to produce sorbitol. In someembodiments, the heat-inactivated lysates are incubated at a temperatureof at least 50 C. In some embodiments, the heat-inactivated lysates areincubated for at least 2 minutes (e.g., at least 3, 4, or 5 minutes).For example, the heat-inactivated lysates may be incubated for 2-5minutes, or 2-10 minutes. The starch may be, for example, amylose,amylopectin, or a mixture of amylose and amylopectin. In someembodiments, biomass is used instead of starch. In these embodiments,the reaction includes cellodextrin phosphorylase(s). In someembodiments, the starch or cellodextrin is present as a component of acompound.

Also provided herein are cells and cell lysates used for the productionof sorbitol. Thus, an engineered cell (e.g., bacterial cell and/or yeastcell) or cell lysate(s) of the present disclosure may include at leastone (e.g., at least two or at least three) enzyme selected from thegroup consisting of α-glucan phosphorylases, phosphoglucomutases, aldosedehydrogenases, and sorbitol-6-phosphate phosphatases. An engineeredcell (e.g., bacterial cell and/or yeast cell) or cell lysate(s) of thepresent disclosure may include at least one (e.g., at least two or atleast three) enzyme selected from the group consisting of cellodextrinphosphorylases, phosphoglucomutases, aldose dehydrogenases, andsorbitol-6-phosphate phosphatases. In some embodiments, an engineeredcell (e.g., bacterial cell and/or yeast cell) or cell lysate(s) of thepresent disclosure includes at least one (e.g., at least two or at leastthree) enzyme selected from the group consisting of thermostableα-glucan phosphorylases, thermostable phosphoglucomutases, thermostablealdose dehydrogenases, and thermostable sorbitol-6-phosphatephosphatases. In some embodiments, an engineered cell (e.g., bacterialcell and/or yeast cell) or cell lysate(s) of the present disclosureincludes at least one (e.g., at least two or at least three) enzymeselected from the group consisting of thermostable cellodextrinphosphorylases, thermostable phosphoglucomutases, thermostable aldosedehydrogenases, and thermostable sorbitol-6-phosphate phosphatases.Thus, an engineered cell (e.g., bacterial cell and/or yeast cell) orcell lysate(s) of the present disclosure may include at least one (e.g.,at least two or at least three) enzyme selected from the groupconsisting of α-glucan phosphorylases, phosphoglucomutases,phosphoglucoisomerases, sorbitol-6-phosphate 2-dehydrogenases, andsorbitol-6-phosphate phosphatases. An engineered cell (e.g., bacterialcell and/or yeast cell) or cell lysate(s) of the present disclosure mayinclude at least one (e.g., at least two or at least three) enzymeselected from the group consisting of cellodextrin phosphorylases,phosphoglucomutases, phosphoglucoisomerases, sorbitol-6-phosphate2-dehydrogenases, and sorbitol-6-phosphate phosphatases. In someembodiments, an engineered cell (e.g., bacterial cell and/or yeast cell)or cell lysate(s) of the present disclosure includes at least one (e.g.,at least two or at least three) enzyme selected from the groupconsisting of thermostable α-glucan phosphorylases, thermostablephosphoglucomutases, thermostable phosphoglucoisomerases, thermostablesorbitol-6-phosphate aldose dehydrogenases, and thermostablesorbitol-6-phosphate phosphatases. In some embodiments, an engineeredcell (e.g., bacterial cell and/or yeast cell) or cell lysate(s) of thepresent disclosure includes at least one (e.g., at least two or at leastthree) enzyme selected from the group consisting of thermostablecellodextrin phosphorylases, thermostable phosphoglucomutases,thermostable phosphoglucoisomerases, thermostable sorbitol-6-phosphate2-dehydrogenases, and thermostable sorbitol-6-phosphate phosphatases.

Ribulose Production

Further aspects of the present disclosure provide methods, compositions,and systems for producing ribulose. These methods, in some embodiments,include culturing cells engineered to express at least one α-glucanphosphorylase and/or cellodextrin phosphorylase, at least onephosphoglucomutase, at least one glucose 6-phosphate dehydrogenase, atleast one 6-phosphogluconolactonase, at least one 6-phosphogluconatedehydrogenase, at least one ribulose 5-phosphate phosphatase, or acombination of at least two of the foregoing enzymes. In someembodiments, the α-glucan phosphorylase (and/or cellodextrinphosphorylase) and the phosphoglucomutase are expressed as a singlefusion (chimeric) protein or a bifunctional protein.

Enzymes of the ribulose production pathways as provided herein aretypically heterologous to the host cell (initially cloned from orobtained from a different cell type), although some of the enzymes maybe endogenous (native) to the host cell. Thus, in some embodiments, atleast one enzyme (e.g., thermostable enzyme) used to convert starchand/or cellodextrin to ribulose is heterologous to the host cell. Insome embodiments, at least two, at least three, at least four, or atleast five enzymes are heterologous to the host cell. In someembodiments, at least one enzyme (e.g., thermostable enzyme) isendogenous (native) to the host cell. In some embodiments, at least two,at least three, at least four, or at least five enzymes are endogenousto the host cell.

The host cells may be prokaryotic cells, such as bacterial cells (e.g.,Escherichia coli cells), or eukaryotic cells, such as yeast cells orplant cells. Other cell types are described below.

In some embodiments, at least one of the enzymes used to convert starchand/or cellodextrin to ribulose is a thermostable enzyme. In someembodiments, at least two, at least three, at least four, or at leastfive of the enzymes are thermostable enzymes. In some embodiments, allof the enzymes are thermostable enzymes. Thus, in some embodiments, themethods include culturing cells engineered to express at least onethermostable α-glucan phosphorylase, at least one thermostablephosphoglucomutase, at least one thermostable glucose 6-phosphatedehydrogenase, at least one thermostable 6-phosphogluconolactonase, atleast one thermostable 6-phosphogluconate dehydrogenase, at least onethermostable ribulose 5-phosphate phosphatase, or a combination of atleast two or more of the foregoing thermostable enzymes. In someembodiments, the methods include culturing cells engineered to expressat least one thermostable cellodextrin phosphorylase, at least onethermostable phosphoglucomutase, at least one thermostable glucose6-phosphate dehydrogenase, at least one thermostable6-phosphogluconolactonase, at least one thermostable 6-phosphogluconatedehydrogenase, at least one thermostable ribulose 5-phosphatephosphatase, or a combination of at least two or more of the foregoingthermostable enzymes.

In some embodiments, the methods of producing ribulose include lysing(e.g., thermal, mechanical, chemical, or enzymatic lysis) the culturedcells to produce at least one (e.g., at least two, at least three, or atleast four) cell lysate. It should be understood that multiple celllysates (and thus multiple cell populations, e.g., from the sameorganism (e.g., bacteria) or from different organisms (e.g., bacteria,yeast and or plant cells)) may be used in an enzymatic reaction asprovided herein. For example, one cell population may be engineered toexpress one or more enzymes(s) of the ribulose production pathway, whileanother cell population (or several other cell populations) may beengineered to express another (at least one other) enzyme of theribulose production pathway. Thus, in some embodiments, the methodscomprise culturing at least one population of cells engineered toexpress at least one α-glucan phosphorylase, culturing at least one cellpopulation engineered to express at least one phosphoglucomutase,culturing at least one cell population engineered to express at leastone glucose 6-phosphate dehydrogenase, culturing at least one cellpopulation engineered to express at least one 6-phosphogluconolactonase,culturing at least one cell population engineered to express at leastone 6-phosphogluconate dehydrogenase, and/or culturing at least one cellpopulation engineered to express at least one ribulose 5-phosphatephosphatase. In some embodiments, the methods comprise culturing atleast one population of cells engineered to express at least onecellodextrin phosphorylase, culturing at least one cell populationengineered to express at least one phosphoglucomutase, culturing atleast one cell population engineered to express at least one glucose6-phosphate dehydrogenase, culturing at least one cell populationengineered to express at least one 6-phosphogluconolactonase, culturingat least one cell population engineered to express at least one6-phosphogluconate dehydrogenase, and/or culturing at least one cellpopulation engineered to express at least one ribulose 5-phosphatephosphatase. Following lysis of the cells, the cell lysates are combinedsuch that the enzymes are present in a single cell lysate/reactionmixture.

In some embodiments, the methods further include heating the celllysate(s) (or a cell lysate mixture) to a temperature that inactivatesundesired native enzymatic activities but does not inactivate any of thethermostable enzymes of the production pathway, to produce aheat-inactivated lysate. The cell lysate(s), in some embodiments, isheated to a temperature of at least 50° C. For example, the celllysate(s) may be heated to a temperature of at least 55° C., 60° C., 65°C., 70° C., 75° C., 80° C., 85° C., or 90° C.

The cell lysate(s) may be heated for a period of time sufficient toinactive native enzymes (or other non-thermostable enzymes) of the cell.For example, the cell lysate(s) may be heated for at least 2, 3, 4, orat least 5 minutes. In some embodiments, the cell lysate(s) are heatedfor longer than 5 minutes. In some embodiments, the cell lysate(s) areheated for a period of time sufficient to reduce the activity of nativeenzymes (or other non-thermostable enzymes) by at least 50% (e.g., atleast 60%, 70%, 80%, or 90%).

Following heat inactivation, in some embodiments, at least one (e.g., atleast two or at least three) purified enzymes is added to the celllysate/reaction mixture. Thus, a reaction mixture, in some embodiments,includes a combination of enzymes present in the cell lysate (expressedby the engineered host cell(s)) and at least one purified enzyme. Atleast one purified enzyme may be selected from the group consisting ofα-glucan phosphorylases or cellodextrin phosphorylases,phosphoglucomutases, glucose 6-phosphate dehydrogenases,6-phosphogluconolactonases, 6-phosphogluconate dehydrogenases, andribulose 5-phosphate phosphatases.

In some embodiments, the methods also include incubating theheat-inactivated lysate(s) in the presence of a starch and inorganicphosphate to produce ribulose. In some embodiments, the methods alsoinclude incubating the heat-inactivated lysate(s) in the presence of acellodextrin and inorganic phosphate to produce ribulose. In someembodiments, the heat-inactivated lysates are incubated at a temperatureof at least 50 C. In some embodiments, the heat-inactivated lysates areincubated for at least 2 minutes (e.g., at least 3, 4, or 5 minutes).For example, the heat-inactivated lysates may be incubated for 2-5minutes, or 2-10 minutes. The starch may be, for example, amylose,amylopectin, or a mixture of amylose and amylopectin. In someembodiments, biomass is used instead of starch. In these embodiments,the reaction includes cellodextrin phosphorylase(s). In someembodiments, the starch or cellodextrin is present as a component of acompound.

Also provided herein are cells and cell lysates used for the productionof ribulose. Thus, an engineered cell (e.g., bacterial cell and/or yeastcell) or cell lysate(s) of the present disclosure may include at leastone (e.g., at least two or at least three) enzyme selected from thegroup consisting of α-glucan phosphorylases, phosphoglucomutases,glucose 6-phosphate dehydrogenases, 6-phosphogluconolactonases,6-phosphogluconate dehydrogenases, and ribulose 5-phosphatephosphatases. An engineered cell (e.g., bacterial cell and/or yeastcell) or cell lysate(s) of the present disclosure may include at leastone (e.g., at least two or at least three) enzyme selected from thegroup consisting of cellodextrin phosphorylases, phosphoglucomutases,glucose 6-phosphate dehydrogenases, 6-phosphogluconolactonases,6-phosphogluconate dehydrogenases, and ribulose 5-phosphatephosphatases. In some embodiments, an engineered cell (e.g., bacterialcell and/or yeast cell) or cell lysate(s) of the present disclosureincludes at least one (e.g., at least two or at least three) enzymeselected from the group consisting of thermostable α-glucanphosphorylases, thermostable phosphoglucomutases, thermostable glucose6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases,thermostable 6-phosphogluconate dehydrogenases, and thermostableribulose 5-phosphate phosphatases. In some embodiments, an engineeredcell (e.g., bacterial cell and/or yeast cell) or cell lysate(s) of thepresent disclosure includes at least one (e.g., at least two or at leastthree) enzyme selected from the group consisting of thermostablecellodextrin phosphorylases, thermostable phosphoglucomutases,thermostable glucose 6-phosphate dehydrogenases, thermostable6-phosphogluconolactonases, thermostable 6-phosphogluconatedehydrogenases, and thermostable ribulose 5-phosphate phosphatases.

Ribose Production

Further still, some aspects of the present disclosure provide methods,compositions, and systems for producing ribose. These methods, in someembodiments, include culturing cells engineered to express at least oneα-glucan phosphorylase and/or at least one cellodextrin phosphorylase,at least one phosphoglucomutase, at least one glucose 6-phosphatedehydrogenase, at least one 6-phosphogluconolactonase, at least one6-phosphogluconate dehydrogenase, at least one ribose 5-phosphateisomerase, at least one ribose 5-phosphate phosphatase, or a combinationof at least two of the foregoing enzymes. In some embodiments, theα-glucan phosphorylase (and/or cellodextrin phosphorylase) and thephosphoglucomutase are expressed as a single fusion (chimeric) proteinor a bifunctional protein.

Enzymes of the ribose production pathways as provided herein aretypically heterologous to the host cell (initially cloned from orobtained from a different cell type), although some of the enzymes maybe endogenous (native) to the host cell. Thus, in some embodiments, atleast one enzyme (e.g., thermostable enzyme) used to convert starchand/or cellodextrin to ribose is heterologous to the host cell. In someembodiments, at least two, at least three, at least four, at least five,or at least six enzymes are heterologous to the host cell. In someembodiments, at least one enzyme (e.g., thermostable enzyme) isendogenous (native) to the host cell. In some embodiments, at least two,at least three, at least four, at least five, or at least six enzymesare endogenous to the host cell.

The host cells may be prokaryotic cells, such as bacterial cells (e.g.,Escherichia coli cells), or eukaryotic cells, such as yeast cells orplant cells. Other cell types are described below.

In some embodiments, at least one of the enzymes used to convert starchand/or cellodextrin to ribose is a thermostable enzyme. In someembodiments, at least two, at least three, at least four, at least five,or at least six of the enzymes are thermostable enzymes. In someembodiments, all of the enzymes are thermostable enzymes. Thus, in someembodiments, the methods include culturing cells engineered to expressat least one thermostable α-glucan phosphorylase, at least onethermostable phosphoglucomutase, at least one thermostable glucose6-phosphate dehydrogenase, at least one thermostable6-phosphogluconolactonase, at least one thermostable 6-phosphogluconatedehydrogenase, at least one thermostable ribose 5-phosphate isomerase,at least one thermostable ribose 5-phosphate phosphatase, or acombination of at least two or more of the foregoing thermostableenzymes. In some embodiments, the methods include culturing cellsengineered to express at least one thermostable cellodextrinphosphorylase, at least one thermostable phosphoglucomutase, at leastone thermostable glucose 6-phosphate dehydrogenase, at least onethermostable 6-phosphogluconolactonase, at least one thermostable6-phosphogluconate dehydrogenase, at least one thermostable ribose5-phosphate isomerase, at least one thermostable ribose 5-phosphatephosphatase, or a combination of at least two or more of the foregoingthermostable enzymes.

In some embodiments, the methods of producing ribose include lysing(e.g., thermal, mechanical, chemical, or enzymatic lysis) the culturedcells to produce at least one (e.g., at least two, at least three, or atleast four) cell lysate. It should be understood that multiple celllysates (and thus multiple cell populations, e.g., from the sameorganism (e.g., bacteria) or from different organisms (e.g., bacteria,yeast and or plant cells)) may be used in an enzymatic reaction asprovided herein. For example, one cell population may be engineered toexpress one or more enzymes(s) of the ribose production pathway, whileanother cell population (or several other cell populations) may beengineered to express another (at least one other) enzyme of the riboseproduction pathway. Thus, in some embodiments, the methods compriseculturing at least one population of cells engineered to express atleast one α-glucan phosphorylase, culturing at least one cell populationengineered to express at least one phosphoglucomutase, culturing atleast one cell population engineered to express at least one glucose6-phosphate dehydrogenase, culturing at least one cell populationengineered to express at least one 6-phosphogluconolactonase, culturingat least one cell population engineered to express at least one6-phosphogluconate dehydrogenase, culturing at least one cell populationengineered to express at least one ribose 5-phosphate isomerase, and/orculturing at least one cell population engineered to express at leastone ribose 5-phosphate phosphatase. In some embodiments, the methodscomprise culturing at least one population of cells engineered toexpress at least one cellodextrin phosphorylase, culturing at least onecell population engineered to express at least one phosphoglucomutase,culturing at least one cell population engineered to express at leastone glucose 6-phosphate dehydrogenase, culturing at least one cellpopulation engineered to express at least one 6-phosphogluconolactonase,culturing at least one cell population engineered to express at leastone 6-phosphogluconate dehydrogenase, culturing at least one cellpopulation engineered to express at least one ribose 5-phosphateisomerase, and/or culturing at least one cell population engineered toexpress at least one ribose 5-phosphate phosphatase. Following lysis ofthe cells, the cell lysates are combined such that the enzymes arepresent in a single cell lysate/reaction mixture.

In some embodiments, the methods further include heating the celllysate(s) (or a cell lysate mixture) to a temperature that inactivatesundesired native enzymatic activities but does not inactivate any of thethermostable enzymes of the production pathway, to produce aheat-inactivated lysate. The cell lysate(s), in some embodiments, isheated to a temperature of at least 50° C. For example, the celllysate(s) may be heated to a temperature of at least 55° C., 60° C., 65°C., 70° C., 75° C., 80° C., 85° C., or 90° C.

The cell lysate(s) may be heated for a period of time sufficient toinactive native enzymes (or other non-thermostable enzymes) of the cell.For example, the cell lysate(s) may be heated for at least 2, 3, 4, orat least 5 minutes. In some embodiments, the cell lysate(s) are heatedfor longer than 5 minutes. In some embodiments, the cell lysate(s) areheated for a period of time sufficient to reduce the activity of nativeenzymes (or other non-thermostable enzymes) by at least 50% (e.g., atleast 60%, 70%, 80%, or 90%).

Following heat inactivation, in some embodiments, at least one (e.g., atleast two or at least three) purified enzymes is added to the celllysate/reaction mixture. Thus, a reaction mixture, in some embodiments,includes a combination of enzymes present in the cell lysate (expressedby the engineered host cell(s)) and at least one purified enzyme. Atleast one purified enzyme may be selected from the group consisting ofα-glucan phosphorylases or cellodextrin phosphorylases,phosphoglucomutases, glucose 6-phosphate dehydrogenases,6-phosphogluconolactonases, 6-phosphogluconate dehydrogenases, ribose5-phosphate isomerases, and ribose 5-phosphate phosphatases.

In some embodiments, the methods also include incubating theheat-inactivated lysate(s) in the presence of a starch and inorganicphosphate to produce ribose. In some embodiments, the methods alsoinclude incubating the heat-inactivated lysate(s) in the presence of acellodextrin and inorganic phosphate to produce ribose. In someembodiments, the heat-inactivated lysates are incubated at a temperatureof at least 50 C. In some embodiments, the heat-inactivated lysates areincubated for at least 2 minutes (e.g., at least 3, 4, or 5 minutes).For example, the heat-inactivated lysates may be incubated for 2-5minutes, or 2-10 minutes. The starch may be, for example, amylose,amylopectin, or a mixture of amylose and amylopectin. In someembodiments, biomass is used instead of starch. In these embodiments,the reaction includes cellodextrin phosphorylase(s). In someembodiments, the starch or cellodextrin is present as a component of acompound.

Also provided herein are cells and cell lysates used for the productionof ribose. Thus, an engineered cell (e.g., bacterial cell and/or yeastcell) or cell lysate(s) of the present disclosure may include at leastone (e.g., at least two, at least three, at least four, at least five,or at least six) enzyme selected from the group consisting of α-glucanphosphorylases, phosphoglucomutases, glucose 6-phosphate dehydrogenases,6-phosphogluconolactonases, 6-phosphogluconate dehydrogenases, ribose5-phosphate isomerases, and ribose 5-phosphate phosphatases. Anengineered cell (e.g., bacterial cell and/or yeast cell) or celllysate(s) of the present disclosure may include at least one (e.g., atleast two, at least three, at least four, at least five, or at leastsix) enzyme selected from the group consisting of cellodextrinphosphorylases, phosphoglucomutases, glucose 6-phosphate dehydrogenases,6-phosphogluconolactonases, 6-phosphogluconate dehydrogenases, ribose5-phosphate isomerases, and ribose 5-phosphate phosphatases. In someembodiments, an engineered cell (e.g., bacterial cell and/or yeast cell)or cell lysate(s) of the present disclosure includes at least one (e.g.,at least two, at least three, at least four, at least five, or at leastsix) enzyme selected from the group consisting of thermostable α-glucanphosphorylases, thermostable phosphoglucomutases, thermostable glucose6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases,thermostable 6-phosphogluconate dehydrogenases, thermostable ribose5-phosphate isomerases, and thermostable ribose 5-phosphatephosphatases. some embodiments, an engineered cell (e.g., bacterial celland/or yeast cell) or cell lysate(s) of the present disclosure includesat least one (e.g., at least two, at least three, at least four, atleast five, or at least six) enzyme selected from the group consistingof thermostable cellodextrin phosphorylases, thermostablephosphoglucomutases, thermostable glucose 6-phosphate dehydrogenases,thermostable 6-phosphogluconolactonases, thermostable 6-phosphogluconatedehydrogenases, thermostable ribose 5-phosphate isomerases, andthermostable ribose 5-phosphate phosphatases.

Arabinose Production

Further still, some aspects of the present disclosure provide methods,compositions, and systems for producing arabinose. These methods, insome embodiments, include culturing cells engineered to express at leastone α-glucan phosphorylase and/or at least one cellodextrinphosphorylase, at least one phosphoglucomutase, at least one glucose6-phosphate dehydrogenase, at least one 6-phosphogluconolactonase, atleast one 6-phosphogluconate dehydrogenase, at least one arabinose5-phosphate isomerase, at least one arabinose 5-phosphate phosphatase,or a combination of at least two of the foregoing enzymes. In someembodiments, the α-glucan phosphorylase (and/or cellodextrinphosphorylase) and the phosphoglucomutase are expressed as a singlefusion (chimeric) protein or a bifunctional protein.

Enzymes of the arabinose production pathways as provided herein aretypically heterologous to the host cell (initially cloned from orobtained from a different cell type), although some of the enzymes maybe endogenous (native) to the host cell. Thus, in some embodiments, atleast one enzyme (e.g., thermostable enzyme) used to convert starchand/or cellodextrin to arabinose is heterologous to the host cell. Insome embodiments, at least two, at least three, at least four, at leastfive, or at least six enzymes are heterologous to the host cell. In someembodiments, at least one enzyme (e.g., thermostable enzyme) isendogenous (native) to the host cell. In some embodiments, at least two,at least three, at least four, at least five, or at least six enzymesare endogenous to the host cell.

The host cells may be prokaryotic cells, such as bacterial cells (e.g.,Escherichia coli cells), or eukaryotic cells, such as yeast cells orplant cells. Other cell types are described below.

In some embodiments, at least one of the enzymes used to convert starchand/or cellodextrin to arabinose is a thermostable enzyme. In someembodiments, at least two, at least three, at least four, at least five,or at least six of the enzymes are thermostable enzymes. In someembodiments, all of the enzymes are thermostable enzymes. Thus, in someembodiments, the methods include culturing cells engineered to expressat least one thermostable α-glucan phosphorylase, at least onethermostable phosphoglucomutase, at least one thermostable glucose6-phosphate dehydrogenase, at least one thermostable6-phosphogluconolactonase, at least one thermostable 6-phosphogluconatedehydrogenase, at least one thermostable arabinose 5-phosphateisomerase, at least one thermostable arabinose 5-phosphate phosphatase,or a combination of at least two or more of the foregoing thermostableenzymes. In some embodiments, the methods include culturing cellsengineered to express at least one thermostable cellodextrinphosphorylase, at least one thermostable phosphoglucomutase, at leastone thermostable glucose 6-phosphate dehydrogenase, at least onethermostable 6-phosphogluconolactonase, at least one thermostable6-phosphogluconate dehydrogenase, at least one thermostable arabinose5-phosphate isomerase, at least one thermostable arabinose 5-phosphatephosphatase, or a combination of at least two or more of the foregoingthermostable enzymes.

In some embodiments, the methods of producing arabinose include lysing(e.g., thermal, mechanical, chemical, or enzymatic lysis) the culturedcells to produce at least one (e.g., at least two, at least three, or atleast four) cell lysate. It should be understood that multiple celllysates (and thus multiple cell populations, e.g., from the sameorganism (e.g., bacteria) or from different organisms (e.g., bacteria,yeast and or plant cells)) may be used in an enzymatic reaction asprovided herein. For example, one cell population may be engineered toexpress one or more enzymes(s) of the arabinose production pathway,while another cell population (or several other cell populations) may beengineered to express another (at least one other) enzyme of thearabinose production pathway. Thus, in some embodiments, the methodscomprise culturing at least one population of cells engineered toexpress at least one α-glucan phosphorylase, culturing at least one cellpopulation engineered to express at least one phosphoglucomutase,culturing at least one cell population engineered to express at leastone glucose 6-phosphate dehydrogenase, culturing at least one cellpopulation engineered to express at least one 6-phosphogluconolactonase,culturing at least one cell population engineered to express at leastone 6-phosphogluconate dehydrogenase, culturing at least one cellpopulation engineered to express at least one arabinose 5-phosphateisomerase, and/or culturing at least one cell population engineered toexpress at least one arabinose 5-phosphate phosphatase. In someembodiments, the methods comprise culturing at least one population ofcells engineered to express at least one cellodextrin phosphorylase,culturing at least one cell population engineered to express at leastone phosphoglucomutase, culturing at least one cell populationengineered to express at least one glucose 6-phosphate dehydrogenase,culturing at least one cell population engineered to express at leastone 6-phosphogluconolactonase, culturing at least one cell populationengineered to express at least one 6-phosphogluconate dehydrogenase,culturing at least one cell population engineered to express at leastone arabinose 5-phosphate isomerase, and/or culturing at least one cellpopulation engineered to express at least one arabinose 5-phosphatephosphatase. Following lysis of the cells, the cell lysates are combinedsuch that the enzymes are present in a single cell lysate/reactionmixture.

In some embodiments, the methods further include heating the celllysate(s) (or a cell lysate mixture) to a temperature that inactivatesundesired native enzymatic activities but does not inactivate any of thethermostable enzymes of the production pathway, to produce aheat-inactivated lysate. The cell lysate(s), in some embodiments, isheated to a temperature of at least 50° C. For example, the celllysate(s) may be heated to a temperature of at least 55° C., 60° C., 65°C., 70° C., 75° C., 80° C., 85° C., or 90° C.

The cell lysate(s) may be heated for a period of time sufficient toinactive native enzymes (or other non-thermostable enzymes) of the cell.For example, the cell lysate(s) may be heated for at least 2, 3, 4, orat least 5 minutes. In some embodiments, the cell lysate(s) are heatedfor longer than 5 minutes. In some embodiments, the cell lysate(s) areheated for a period of time sufficient to reduce the activity of nativeenzymes (or other non-thermostable enzymes) by at least 50% (e.g., atleast 60%, 70%, 80%, or 90%).

Following heat inactivation, in some embodiments, at least one (e.g., atleast two or at least three) purified enzymes is added to the celllysate/reaction mixture. Thus, a reaction mixture, in some embodiments,includes a combination of enzymes present in the cell lysate (expressedby the engineered host cell(s)) and at least one purified enzyme. Atleast one purified enzyme may be selected from the group consisting ofα-glucan phosphorylases or cellodextrin phosphorylases,phosphoglucomutases, glucose 6-phosphate dehydrogenases,6-phosphogluconolactonases, 6-phosphogluconate dehydrogenases, arabinose5-phosphate isomerases, and arabinose 5-phosphate phosphatases.

In some embodiments, the methods also include incubating theheat-inactivated lysate(s) in the presence of a starch and inorganicphosphate to produce arabinose. In some embodiments, the methods alsoinclude incubating the heat-inactivated lysate(s) in the presence of acellodextrin and inorganic phosphate to produce arabinose. In someembodiments, the heat-inactivated lysates are incubated at a temperatureof at least 50 C. In some embodiments, the heat-inactivated lysates areincubated for at least 2 minutes (e.g., at least 3, 4, or 5 minutes).For example, the heat-inactivated lysates may be incubated for 2-5minutes, or 2-10 minutes. The starch may be, for example, amylose,amylopectin, or a mixture of amylose and amylopectin. In someembodiments, biomass is used instead of starch. In these embodiments,the reaction includes cellodextrin phosphorylase(s). In someembodiments, the starch or cellodextrin is present as a component of acompound.

Also provided herein are cells and cell lysates used for the productionof arabinose. Thus, an engineered cell (e.g., bacterial cell and/oryeast cell) or cell lysate(s) of the present disclosure may include atleast one (e.g., at least two, at least three, at least four, at leastfive, or at least six) enzyme selected from the group consisting ofα-glucan phosphorylases, phosphoglucomutases, glucose 6-phosphatedehydrogenases, 6-phosphogluconolactonases, 6-phosphogluconatedehydrogenases, arabinose 5-phosphate isomerases, and arabinose5-phosphate phosphatases. An engineered cell (e.g., bacterial celland/or yeast cell) or cell lysate(s) of the present disclosure mayinclude at least one (e.g., at least two, at least three, at least four,at least five, or at least six) enzyme selected from the groupconsisting of cellodextrin phosphorylases, phosphoglucomutases, glucose6-phosphate dehydrogenases, 6-phosphogluconolactonases,6-phosphogluconate dehydrogenases, arabinose 5-phosphate isomerases, andarabinose 5-phosphate phosphatases. In some embodiments, an engineeredcell (e.g., bacterial cell and/or yeast cell) or cell lysate(s) of thepresent disclosure includes at least one (e.g., at least two, at leastthree, at least four, at least five, or at least six) enzyme selectedfrom the group consisting of thermostable α-glucan phosphorylases,thermostable phosphoglucomutases, thermostable glucose 6-phosphatedehydrogenases, thermostable 6-phosphogluconolactonases, thermostable6-phosphogluconate dehydrogenases, thermostable arabinose 5-phosphateisomerases, and thermostable arabinose 5-phosphate phosphatases. In someembodiments, an engineered cell (e.g., bacterial cell and/or yeast cell)or cell lysate(s) of the present disclosure includes at least one (e.g.,at least two, at least three, at least four, at least five, or at leastsix) enzyme selected from the group consisting of thermostablecellodextrin phosphorylases, thermostable phosphoglucomutases,thermostable glucose 6-phosphate dehydrogenases, thermostable6-phosphogluconolactonases, thermostable 6-phosphogluconatedehydrogenases, thermostable arabinose 5-phosphate isomerases, andthermostable arabinose 5-phosphate phosphatases.

Substrate Flexibility and Debranching Enzymes

For all pathways described herein, a multitude of polymeric glucosesubstrates can be used. Non-limiting examples of polymeric glucosesubstrates include starch, glycogen, and cellodextrin. In someembodiments, the substrate is starch. In other embodiments, thesubstrate is glycogen. In still other embodiments, the substrate iscellodextrin. In some embodiments, a partially hydrolyzed version of apolymeric glucose substrate (e.g., starch, glycogen, orcellulose/cellodextrin) is used. Starch and glycogen include a pluralityof glucose monomers linked primarily by α(1-4) bonds, while cellodextrinincludes the same glucose monomers linked by β(1-4) bonds. One othermajor difference between cellodextrin and the other two substrates isthe existence of α(1-6) branches off of the α(1-4) chains. Both starchand glycogen contain these branch points, although glycogen issubstantially more branched than starch. For the α(1-4) polymers,α-glucan phosphorylases, also referred to as α-glucan phosphorylases orglycogen phosphorylases depending on substrate preference, consume thepolymers one glucose at a time releasing glucose 1-phosphate. Forcellodextrin, cellodextrin phosphorylase performs the same reaction,also releasing glucose 1-phosphate.

Long polymers of starch and cellulose/cellodextrin are often insolublein aqueous solutions and in addition to precipitating out, can causegelling and retrogradation of the solution. When starch andcellulose/cellodextrin are partially hydrolyzed to smaller chain lengthpolymers, either through chemical (e.g., acid hydrolysis) or enzymatic(e.g., α-amylase) methods, the resulting products are maltodextrins andcellodextrins for starch and cellulose, respectively. These hydrolyzedderivatives often solubilize and mix better than their parent molecules,and thus, in some embodiments, are used in the pathways provided herein.

For glycogen, starch, or hydrolyzed maltodextrins, α(1-6) branches willsubstantially reduce yields of any sugar pathway, as the glucanphosphorylase chew the polymers down to the end of their branches,leaving a large central core of available glucose unconverted. For thesesubstrates/pathways, debranching enzymes may be used to increasesubstrate availability to the glucan phosphorylase. There are twoexemplary classes of debranching enzymes that can be used—isoamylasesand pullulanases (see, e.g., Table 3). Enzymatically, both classesperform the same function but differ in substrate specificity. Whileusing the debranching enzyme increases yields, the timing of the usewill depend on the process and substrates being used. In someembodiments, an α-glucan is pretreated with α-amylase and a debranchingenzyme, and then the resulting debranched maltodextrin(s) is fed into areactor with the other pathway enzymes. In other embodiments, thedebranching occurs concurrent with the pathway and branched α-glucans isfed into the reaction containing all pathway enzymes as well as thedebranching enzyme.

TABLE 3 Exemplary Debranching Enzymes Enzyme Name EC No. Native OrganismNCBI No. Pullulanase 3.2.1.41 Fervidobacterium AAD30387 pennavoransThermotoga sp. RQ5 WP_012310857 Bacillus flavocaldarius BAB18516Thermosipho africanus WP_004100450 Kosmotoga olearia WP_015868997Isoamylase 3.2.1.68 Sulfolobus tokodaii BAB65940 Metallosphaerahakonensis AAS00512 Sphaerobacter thermophilus WP_012873143 Bacilluslentus AGL34022Cell-Free Production

“Cell-free production” is the use of biological processes for thesynthesis of a biomolecule or chemical compound without using livingcells. Rather, the cells are lysed and unpurified (crude) portions,containing enzymes, are used for the production of a desired product. Asa non-limiting example, cells are cultured, harvested, and lysed byhigh-pressure homogenization. The cell-free reaction may be conducted ina batch or fed-batch mode. In some instances, the biological reactionnetworks fill the working volume of the reactor and may be more dilutethan the intracellular environment. Yet substantially all of thecellular catalysts are provided, including catalysts that are membraneassociated. The inner membrane is fragmented during cell lysis, and thefragments of these membranes form functional membrane vesicles. Thus,complex biotransformations are effected by catalysis. See, e.g., Swartz,AIChE Journal, 2012, 58(1), 5-13, incorporated herein by reference.

Cell-free methods and systems of the present disclosure, in someembodiments, utilize cell lysates (e.g., crude or partially purifiedcell lysates), discussed in greater detail herein. Cell lysates may beprepared, for example, by mechanical means (e.g., shearing or crushing).In some embodiments, cell lysates are distinct fromchemically-permeabilized cells. As discussed here, in some embodiments,during cell lysis (e.g., mechanical cell lysis), the inner cell membraneis fragmented such that inverted membrane vesicles are formed in thecells lysates. Cells that are lysed (e.g., at least 75%, 80%, 85%, 90%,or 95%) are no longer intact.

In some embodiments, permeabilized cells are used. Permeabilized cellsare intact cells containing perforations (small holes). In someembodiments, cells may be permeabilized to release the cell content foruse in a reaction as provided herein.

In some embodiments, partially purified cell fractions are used. Apartially purified cell fraction is a cell lysate from which one or morecellular components (e.g., cell membranes) have been partially orcompletely removed.

Thermostable Enzymes

An enzyme is considered thermostable if the enzyme (a) retains asubstantial portion of its activity after exposure to high temperaturesthat denature other native enzymes or (b) functions at a relatively highrate after exposure to a medium to high temperature where native enzymesfunction at low rates.

In some embodiments, a thermostable enzyme retains greater than 50%activity following exposure to relatively high temperature that wouldotherwise denature a similar (non-thermostable) native enzyme. In someembodiments, a thermostable enzyme retains 50-100% activity followingexposure to relatively high temperature that would otherwise denature asimilar (non-thermostable) native enzyme. For example, a thermostableenzyme may retain 50-90%, 50-85%, 50-80%, 50-75%, 50-70%, 50-65%,50-60%, or 50-55% of its activity following exposure to relatively hightemperature that would otherwise denature a similar (non-thermostable)native enzyme. In some embodiments, a thermostable enzyme retains 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, 99%, or 100% of its activity following exposure to relatively hightemperature that would otherwise denature a similar (non-thermostable)native enzyme.

In some embodiments, the activity of a thermostable enzyme afterexposure medium to high temperature is greater than (e.g., 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%,99%, or 100% greater than) the activity of a similar (non-thermostable)native enzyme.

Thermostable enzymes (e.g., phosphatases or phosphorylases) may remainactive (able to catalyze a reaction), for example, at temperatures of45° C. to 80° C., or higher. In some embodiments, thermostable enzymesremain active at a temperature of 45-80° C., 45-70° C., 45-60° C.,45-50° C., 50-80° C., 50-70° C., 50-60° C., 60-80° C., 60-70° C., or70-80° C. For example, thermostable enzymes may remain active at atemperature of 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C.,52° C., 53° C., 54° C., 55° C., 55° C., 56° C., 57° C., 58° C., 59° C.,60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C.,69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77° C.,78° C., 79° C., or 80° C. Thermostable enzymes may remain active atrelatively high temperatures for 15 minutes to 48 hours, or longer,after exposure to relatively high temperatures. For example,thermostable enzymes may remain active at relatively high temperaturesfor 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 24, 36, 42, or 48 hours.

Engineered Cells

Engineered cells of the present disclosure, in some embodiments,comprise at least one, or all, of the enzymatic activities required toconvert a starch and/or cellulose/cellodextrin to a sugar. “Engineeredcells” are cells that comprise at least one engineered (e.g.,recombinant or synthetic) nucleic acid, or are otherwise modified suchthat they are structurally and/or functionally distinct from theirnaturally-occurring counterparts. Thus, a cell that contains anengineered nucleic acid is considered an “engineered cell.”

Engineered cells of the present disclosure, in some embodiments,comprise a α-glucan phosphorylase (e.g., a thermostable α-glucanphosphorylase) and/or a cellodextrin phosphorylase (e.g., a thermostablecellodextrin phosphorylase), a phosphoglucomutase (e.g., a thermostablephosphoglucomutase), and at least one enzyme (e.g., thermostable enzyme)selected from the group consisting of isomerases, epimerases,dehydrogenases, and sugar phosphatases.

Engineered cells, in some embodiments, express selectable markers.Selectable markers are typically used to select engineered cells thathave taken up and express an engineered nucleic acid followingtransfection of the cell (or following other procedure used to introduceforeign nucleic acid into the cell). Thus, a nucleic acid encodingproduct may also encode a selectable marker. Examples of selectablemarkers include, without limitation, genes encoding proteins thatincrease or decrease either resistance or sensitivity to antibiotics(e.g., ampicillin resistance genes, kanamycin resistance genes, neomycinresistance genes, tetracycline resistance genes and chloramphenicolresistance genes) or other compounds. Other selectable markers may beused in accordance with the present disclosure.

An engineered cell “expresses” a product if the product, encoded by anucleic acid (e.g., an engineered nucleic acid), is produced in thecell. It is known in the art that gene expression refers to the processby which genetic instructions in the form of a nucleic acid are used tosynthesize a product, such as a protein (e.g., an enzyme).

Engineered cells may be prokaryotic cells or eukaryotic cells. In someembodiments, engineered cells are bacterial cells, yeast cells, insectcells, mammalian cells, or other types of cells.

Engineered bacterial cells useful in the present disclosure include,without limitation, engineered Escherichia spp., Streptomyces spp.,Zymonas spp., Acetobacter spp., Citrobacter spp., Synechocystis spp.,Rhizobium spp., Clostridium spp., Corynebacterium spp., Streptococcusspp., Xanthomonas spp., Lactobacillus spp., Lactococcus spp., Bacillusspp., Alcaligenes spp., Pseudomonas spp., Aeromonas spp., Azotobacterspp., Comamonas spp., Mycobacterium spp., Rhodococcus spp.,Gluconobacter spp., Ralstonia spp., Acidithiobacillus spp., Microlunatusspp., Geobacter spp., Geobacillus spp., Arthrobacter spp.,Flavobacterium spp., Serratia spp., Saccharopolyspora spp., Thermusspp., Stenotrophomonas spp., Chromobacterium spp., Sinorhizobium spp.,Saccharopolyspora spp., Agrobacterium spp., Vibrio spp., and Pantoeaspp.

Engineered yeast cells useful in the present disclosure include, withoutlimitation, engineered Saccharomyces spp., Schizosaccharomyces,Hansenula, Candida, Kluyveromyces, Yarrowia and Pichia.

In some embodiments, engineered cells useful in the present disclosureare engineered Escherichia coli cells, Bacillus subtilis cells,Pseudomonas putida cells, Saccharomyces cerevisiae cells, and/orLactobacillus brevis cells. In some embodiments, engineered cells usefulin the present disclosure are engineered Escherichia coli cells.

Engineered Nucleic Acids

A “nucleic acid” is at least two nucleotides covalently linked together,and in some instances, may contain phosphodiester bonds (e.g., aphosphodiester “backbone”). Nucleic acids (e.g., components, orportions, of nucleic acids) may be naturally occurring or engineered.“Naturally occurring” nucleic acids are present in a cell that exists innature in the absence of human intervention. “Engineered nucleic acids”include recombinant nucleic acids and synthetic nucleic acids. A“recombinant nucleic acid” refers to a molecule that is constructed byjoining nucleic acid molecules (e.g., from the same species or fromdifferent species) and, typically, can replicate in a living cell. A“synthetic nucleic acid” refers to a molecule that is biologicallysynthesized, chemically synthesized, or by other means synthesized oramplified. A synthetic nucleic acid includes nucleic acids that arechemically modified or otherwise modified but can base pair withnaturally-occurring nucleic acid molecules. Recombinant and syntheticnucleic acids also include those molecules that result from thereplication of either of the foregoing. Engineered nucleic acids maycontain portions of nucleic acids that are naturally occurring, but as awhole, engineered nucleic acids do not occur naturally and require humanintervention. In some embodiments, a nucleic acid encoding a product ofthe present disclosure is a recombinant nucleic acid or a syntheticnucleic acid. In other embodiments, a nucleic acid encoding a product isnaturally occurring.

An engineered nucleic acid encoding enzymes, as provided herein, may beoperably linked to a “promoter,” which is a control region of a nucleicacid at which initiation and rate of transcription of the remainder of anucleic acid are controlled. A promoter drives expression or drivestranscription of the nucleic acid that it regulates.

A promoter may be one naturally associated with a gene or sequence, asmay be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment of a given gene or sequence. Such apromoter can be referred to as “endogenous.”

In some embodiments, a coding nucleic acid sequence may be positionedunder the control of a recombinant or heterologous promoter, whichrefers to a promoter that is not normally associated with the encodedsequence in its natural environment. Such promoters may includepromoters of other genes; promoters isolated from any other cell; andsynthetic promoters or enhancers that are not “naturally occurring” suchas, for example, those that contain different elements of differenttranscriptional regulatory regions and/or mutations that alterexpression through methods of genetic engineering that are known in theart. In addition to producing nucleic acid sequences of promoters andenhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, includingpolymerase chain reaction (PCR).

A promoter is considered to be “operably linked” when it is in a correctfunctional location and orientation in relation to the nucleic acid itregulates to control (“drive”) transcriptional initiation and/orexpression of that nucleic acid.

Engineered nucleic acids of the present disclosure may contain aconstitutive promoter or an inducible promoter. A “constitutivepromoter” refers to a promoter that is constantly active in a cell. An“inducible promoter” refers to a promoter that initiates or enhancestranscriptional activity when in the presence of, influenced by, orcontacted by an inducer or inducing agent, or activated in the absenceof a factor that causes repression. Inducible promoters for use inaccordance with the present disclosure include any inducible promoterdescribed herein or known to one of ordinary skill in the art. Examplesof inducible promoters include, without limitation,chemically/biochemically-regulated and physically-regulated promoterssuch as alcohol-regulated promoters, tetracycline-regulated promoters,steroid-regulated promoters, metal-regulated promoters,pathogenesis-regulated promoters, temperature/heat-inducible,phosphate-regulated (e.g., PhoA), and light-regulated promoters.

An inducer or inducing agent may be endogenous or a normally exogenouscondition (e.g., light), compound (e.g., chemical or non-chemicalcompound) or protein that contacts an inducible promoter in such a wayas to be active in regulating transcriptional activity from theinducible promoter. Thus, a “signal that regulates transcription” of anucleic acid refers to an inducer signal that acts on an induciblepromoter. A signal that regulates transcription may activate orinactivate transcription, depending on the regulatory system used.Activation of transcription may involve directly acting on a promoter todrive transcription or indirectly acting on a promoter by inactivation arepressor that is preventing the promoter from driving transcription.Conversely, deactivation of transcription may involve directly acting ona promoter to prevent transcription or indirectly acting on a promoterby activating a repressor that then acts on the promoter.

Engineered nucleic acids may be introduced into host cells using anymeans known in the art, including, without limitation, transformation,transfection (e.g., chemical (e.g., calcium phosphate, cationicpolymers, or liposomes) or non-chemical (e.g., electroporation,sonoporation, impalefection, optical transfection, hydro dynamic)), andtransduction (e.g., viral transduction).

Enzymes or other proteins encoded by a naturally-occurring,intracellular nucleic acid may be referred to as “endogenous enzymes” or“endogenous proteins.”

Protease Targeting

Engineered cells of the present disclosure may express (e.g.,endogenously express) enzymes necessary for the health of the cells thatmay have a negative impact on the production of a sugar of interest(e.g., allulose). Such enzymes are referred to herein as “targetenzymes.” For example, target enzymes expressed by engineered cells maycompete for substrates or cofactors with an enzyme that increases therate of precursor supplied to an sugar production pathway. As anotherexample, target enzymes expressed by the engineered cells may competefor substrates or cofactors with an enzyme that is a key pathway entryenzyme of an sugar production pathway. As yet another example, targetenzymes expressed by the engineered cells may compete for substrates orcofactors with an enzyme that supplies a substrate or cofactor of ansugar production pathway.

To negate, or reduce, this negative impact, target enzymes can bemodified to include a site-specific protease-recognition sequence intheir protein sequence such that the target enzyme may be “targeted” andcleaved for inactivation during sugar production (see, e.g., U.S.Publication No. 2012/0052547 A1, published on Mar. 1, 2012; andInternational Publication No. WO 2015/021058 A2, published Feb. 12,2015, each of which is incorporated by reference herein).

Cleavage of a target enzyme containing a site-specificprotease-recognition sequence results from contact with a cognatesite-specific protease that is sequestered in the periplasm of cell(separate from the target enzyme) during the cell growth phase (e.g., asengineered cells are cultured) and is brought into contact with thetarget enzyme during the conversion phase (e.g., following cell lysis toproduce a cell lysate). Thus, engineered cells of the present disclosurecomprise, in some embodiments, (i) an engineered nucleic acid encoding atarget enzyme that negatively impacts the rate of conversion andincludes a site-specific protease-recognition sequence in the proteinsequence of the target enzyme, and (ii) an engineered nucleic acidencoding a site-specific protease that cleaves the site-specificprotease-recognition sequence of the target enzyme and includes aperiplasmic-targeting sequence. This periplasmic-targeting sequence isresponsible for sequestering the site-specific protease to theperiplasmic space of the cell until the cell is lysed. Examples ofperiplasmic-targeting sequences are provided below.

Examples of proteases that may be used in accordance with the presentdisclosure include, without limitation, alanine carboxypeptidase,astacin, bacterial leucyl aminopeptidase, cancer procoagulant, cathepsinB, clostripain, cytosol alanyl aminopeptidase, elastase, endoproteinaseBrg-C, enterokinase, gastricsin, gelatinase, Gly-X carboxypeptidase,glycyl endopeptidase, human rhinovirus 3C protease, hypodermin C,Iga-specific serine endopeptidase, leucyl aminopeptidase, leucylendopeptidase, lysC, lysosomal pro-X carboxypeptidase, lysylaminopeptidase, methionyl aminopeptidase, myxobacter, nardilysin,pancreatic endopeptidase E, picornain 2B, picornain 3C,proendopeptidase, prolyl aminopeptidase, proprotein convertase I,proprotein convertase II, russellysin, saccharopepsin, semenogelase,T-plasminogen activator, thrombin, tissue kallikrein, tobacco etch virus(TEV), togavirin, tryptophanyl aminopeptidase, U-plasminogen activator,V8, venombin B, venombin BB and Xaa-pro aminopeptidase.

Periplasmic Targeting

Enzymes of an sugar production pathway may include at least one enzymethat has a negative impact on the health (e.g., viability) of a cell. Tonegate or reduce this negative impact, an enzyme can be modified toinclude a relocation sequence such that the enzyme is relocated to acellular or extra-cellular compartment where it is not naturally locatedand where the enzyme does not negatively impact the health of the cell(see, e.g., Publication No. US-2011-0275116-A1, published on Nov. 10,2011, incorporated by reference herein). For example, an enzyme of ansugar production pathway may be relocated to the periplasmic space of acell.

Thus, in some embodiments, engineered cells of the present disclosurecomprise at least one enzyme of an sugar production pathway that islinked to a periplasmic-targeting sequence. A “periplasmic-targetingsequence” is an amino acid sequence that targets to the periplasm of acell the protein to which it is linked. A protein that is linked to aperiplasmic-targeting sequence will be sequestered in the periplasm ofthe cell in which the protein is expressed.

Periplasmic-targeting sequences may be derived from the N-terminus ofbacterial secretory protein, for example. The sequences vary in lengthfrom about 15 to about 70 amino acids. The primary amino acid sequencesof periplasmic-targeting sequences vary, but generally have a commonstructure, including the following components: (i) the N-terminal parthas a variable length and generally carries a net positive charge; (ii)following is a central hydrophobic core of about 6 to about 15 aminoacids; and (iii) the final component includes four to six amino acidswhich define the cleavage site for signal peptidases.

Periplasmic-targeting sequences of the present disclosure, in someembodiments, may be derived from a protein that is secreted in a Gramnegative bacterium. The secreted protein may be encoded by thebacterium, or by a bacteriophage that infects the bacterium. Examples ofGram negative bacterial sources of secreted proteins include, withoutlimitation, members of the genera Escherichia, Pseudomonas, Klebsiella,Salmonella, Caulobacter, Methylomonas, Acetobacter, Achromobacter,Acinetobacter, Aeromonas, Agrobacterium, Alcaligenes, Azotobacter,Burkholderia, Citrobacter, Comamonas, Enterobacter, Erwinia, Rhizobium,Vibrio, and Xanthomonas.

Examples of periplasmic-targeting sequences for use in accordance withthe present disclosure include, without limitation, sequences selectedfrom the group consisting of:

(SEQ ID NO: 1) MKIKTGARILALSALTTMMFSASALA; (SEQ ID NO: 2)MKQSTIALALLPLLFTPVTKA; (SEQ ID NO: 3) MMITLRKLPLAVAVAAGVMSAQAMA;(SEQ ID NO: 4) MNKKVLTLSAVMASMLFGAAAHA; (SEQ ID NO: 5)MKYLLPTAAAGLLLLAAQPAMA; (SEQ ID NO: 6) MKKIWLALAGLVLAFSASA;(SEQ ID NO: 7) MMTKIKLLMLIIFYLIISASAHA; (SEQ ID NO: 8)MKQALRVAFGFLILWASVLHA; (SEQ ID NO: 9) MRVLLFLLLSLFMLPAFS; and(SEQ ID NO: 10) MANNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA.Cell Cultures and Cell Lysates

Typically, engineered cells are cultured. “Culturing” refers to theprocess by which cells are grown under controlled conditions, typicallyoutside of their natural environment. For example, engineered cells,such as engineered bacterial cells, may be grown as a cell suspension inliquid nutrient broth, also referred to as liquid “culture medium.”

In some embodiments, unconverted starch is used as a substrate feed forgrowing cells.

Examples of commonly used bacterial Escherichia coli growth mediainclude, without limitation, LB (Luria Bertani) Miller broth (1% NaCl):1% peptone, 0.5% yeast extract, and 1% NaCl; LB (Luria Bertani) LennoxBroth (0.5% NaCl): 1% peptone, 0.5% yeast extract, and 0.5% NaCl; SOBmedium (Super Optimal Broth): 2% peptone, 0.5% Yeast extract, 10 mMNaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4; SOC medium (Super Optimalbroth with Catabolic repressor): SOB+20 mM glucose; 2× YT broth (2×Yeast extract and Tryptone): 1.6% peptone, 1% yeast extract, and 0.5%NaCl; TB (Terrific Broth) medium: 1.2% peptone, 2.4% yeast extract, 72mM K2HPO4, 17 mM KH2PO4 and 0.4% glycerol; and SB (Super Broth) medium:3.2% peptone, 2% yeast extract, and 0.5% NaCl and or Korz medium (Korz,D J et al. 1995).

Examples of high density bacterial Escherichia coli growth mediainclude, but are not limited to, DNAGro™ medium, ProGro™ medium, AutoX™medium, DetoX™ medium, InduX™ medium, and SecPro™ medium.

In some embodiments, engineered cells are cultured under conditions thatresult in expression of enzymes or nucleic acids. Such cultureconditions may depend on the particular product being expressed and thedesired amount of the product.

In some embodiments, engineered cells are cultured at a temperature of30° C. to 40° C. For example, engineered cells may be cultured at atemperature of 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C.,37° C., 38° C., 39° C. or 40° C. Typically, engineered cells, such asengineered bacterial cells, are cultured at a temperature of 37° C.

In some embodiments, engineered cells are cultured for a period of timeof 12 hours to 72 hours, or more. For example, engineered cells may becultured for a period of time of 12, 18, 24, 30, 36, 42, 48, 54, 60, 66,or 72 hours. Typically, engineered cells, such as engineered bacterialcells, are cultured for a period of time of 12 to 24 hours. In someembodiments, engineered cells are cultured for 12 to 24 hours at atemperature of 37° C.

In some embodiments, engineered cells are cultured (e.g., in liquid cellculture medium) to an optical density, measured at a wavelength of 600nm (OD600), of 5 to 25. In some embodiments, engineered cells arecultured to an OD600 of 5, 10, 15, 20, or 25.

In some embodiments, engineered cells are cultured to a density of 1×10⁴to 1×10⁸ viable cells/ml cell culture medium. In some embodiments,engineered cells are cultured to a density of 1×10⁴, 2×10⁴, 3×10⁴,4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵,5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶,6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷,7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 1×10⁹, or 1×10¹⁰ viable cells/ml. In someembodiments, engineered cells are cultured to a density of 1×10⁸ to1×10¹⁰ viable cells/ml. In some embodiments, engineered cells arecultured to a density of 2×10⁵ to 3×10⁷ viable cells/ml.

In some embodiments, engineered cells are cultured in a bioreactor. Abioreactor refers simply to a container in which cells are cultured,such as a culture flask, a dish, or a bag that may be single-use(disposable), autoclavable, or sterilizable. The bioreactor may be madeof glass, or it may be polymer-based, or it may be made of othermaterials.

Examples of bioreactors include, without limitation, stirred tank (e.g.,well mixed) bioreactors and tubular (e.g., plug flow) bioreactors,airlift bioreactors, membrane stirred tanks, spin filter stirred tanks,vibromixers, fluidized bed reactors, and membrane bioreactors. The modeof operating the bioreactor may be a batch or continuous processes andwill depend on the engineered cells being cultured. A bioreactor iscontinuous when the feed and product streams are continuously being fedand withdrawn from the system. A batch bioreactor may have a continuousrecirculating flow, but no continuous feeding of nutrient or productharvest. For intermittent-harvest and fed-batch (or batch fed) cultures,cells are inoculated at a lower viable cell density in a medium that issimilar in composition to a batch medium. Cells are allowed to growexponentially with essentially no external manipulation until nutrientsare somewhat depleted and cells are approaching stationary growth phase.At this point, for an intermittent harvest batch-fed process, a portionof the cells and product may be harvested, and the removed culturemedium is replenished with fresh medium. This process may be repeatedseveral times. For production of recombinant proteins and antibodies, afed-batch process may be used. While cells are growing exponentially,but nutrients are becoming depleted, concentrated feed medium (e.g.,10-15 times concentrated basal medium) is added either continuously orintermittently to supply additional nutrients, allowing for furtherincrease in cell concentration and the length of the conversion phase.Fresh medium may be added proportionally to cell concentration withoutremoval of culture medium (broth). To accommodate the addition ofmedium, a fed-batch culture is started in a volume much lower that thefull capacity of the bioreactor (e.g., approximately 40% to 50% of themaximum volume).

Some methods of the present disclosure are directed to large-scaleproduction of sugar. For large-scale production methods, engineeredcells may be grown in liquid culture medium in a volume of 5 liters (L)to 50 L, or more. In some embodiments, engineered cells may be grown inliquid culture medium in a volume of greater than (or equal to) 10 L. Insome embodiments, engineered cells are grown in liquid culture medium ina volume of 5 L, 10 L, 15 L, 20 L, 25 L, 30 L, 35 L, 40 L, 45 L, 50 L,or more. In some embodiments, engineered cells may be grown in liquidculture medium in a volume of 5 L to 10 L, 5 L to 15 L, 5 L to 20 L, 5 Lto 25 L, 5 L to 30 L, 5 L to 35 L, 5 L to 40 L, 5 L to 45 L, 10 L to 15L, 10 L to 20 L, 10 L to 25 L, 20 L to 30 L, 10 L to 35 L, 10 L to 40 L,10 L to 45 L, 10 L to 50 L, 15 L to 20 L, 15 L to 25 L, 15 L to 30 L, 15L to 35 L, 15 L to 40 L, 15 L to 45 L, or 15 to 50 L.

Typically, culturing of engineered cells is followed by lysing thecells. “Lysing” refers to the process by which cells are broken down,for example, by viral, enzymatic, mechanical, chemical, heat or osmoticmechanisms. A “cell lysate” refers to a fluid containing the contents oflysed cells (e.g., lysed engineered cells), including, for example,organelles, membrane lipids, proteins, nucleic acids and invertedmembrane vesicles. Cell lysates of the present disclosure may beproduced by lysing any population of engineered cells, as providedherein. A “cell lysate” may exclude permeabilized/perforated cells.

Methods of cell lysis, referred to as “lysing,” are known in the art,any of which may be used in accordance with the present disclosure. Suchcell lysis methods include, without limitation, physical/mechanicallysis, such as homogenization, as well as chemical, thermal, and/orenzymatic lysis.

Cell lysis can disturb carefully controlled cellular environments,resulting in protein degradation and modification by unregulatedendogenous proteases and phosphatases. Thus, in some embodiments,protease inhibitors and/or phosphatase inhibitors may be added to thecell lysate or cells before lysis, or these activities may be removed bygene inactivation or protease targeting.

Cell lysates, in some embodiments, may be combined with at least onenutrient. For example, cell lysates may be combined with Na₂HPO₄,KH₂PO₄, NH₄Cl, NaCl, MgSO₄, CaCl₂. Examples of other nutrients include,without limitation, magnesium sulfate, magnesium chloride, magnesiumorotate, magnesium citrate, potassium phosphate monobasic, potassiumphosphate dibasic, potassium phosphate tribasic, sodium phosphatemonobasic, sodium phosphate dibasic, sodium phosphate tribasic, ammoniumphosphate monobasic, ammonium phosphate dibasic, ammonium sulfate,ammonium chloride, ammonium hydroxide,

In some embodiments, cell lysates may consist of disrupted cellsuspensions that are further modified by chemical, thermal, enzymatic ormechanical means to enrich or purify or reduce or eliminate specificcomponents. For example, following disruption via mechanical, thermal,chemical or enzymatic means, as described above, the resulting materialmay be subjected to mechanical separation, e.g. membrane filtration,centrifugation or others, to partially enrich for a select enzymaticactivity or to eliminate an undesired enzymatic activity or lysatecomponent. Further examples may include the addition of salts orsolvents to a disrupted cell suspension or alteration of the pH ortemperature of the disrupted cell suspension resulting in theprecipitation of desired activities followed by mechanical separation ofthese precipitated components as described above. Conversely, theaddition of salts or solvents or the alteration of pH or temperature canbe leveraged to eliminate undesired activities through eitherinactivation of those enzymes or precipitation and subsequent mechanicalseparation of the undesired enzymatic activity or activities.

Cell lysates, in some embodiments, may be combined with at least onecofactor. For example, cell lysates may be combined with adenosinediphosphate (ADP), adenosine triphosphate (ATP), nicotinamide adeninedinucleotide (NAD+), or other non-protein chemical compounds requiredfor activity of an enzyme (e.g., inorganic ions and coenzymes).

In some embodiments, cell lysates are incubated under conditions thatresult in conversion of starch or cellulose/cellodextrin to sugar.

The volume of cell lysate used for a single reaction may vary. In someembodiments, the volume of a cell lysate is 1 to 150 m³. For example,the volume of a cell lysate may be 1 m³, 5 m³, 10 m³, 15 m³, 20 m³, 25m³, 30 m³, 35 m³, 40 m³, 45 m³, 50 m³, 55 m³, 60 m³, 65 m³, 70 m³, 75m³, 80 m³, 85 m³, 90 m³, 95 m³, 100 m³, 105 m³, 110 m³, 115 m³, 120 m³,125 m³, 130 m³, 135 m³, 140 m³, 145 m³, or 150 m³. In some embodiments,the volume of a cell lysate is 25 m³ to 150 m³, 50 m³ to 150 m³, or 100m³ to 150 m³.

Purified Enzymes

In some embodiments of the present invention enzymes may be purifiedprior to addition to the production reaction. Enzyme purification shouldbe understood to mean any enrichment or extraction of a specific enzymeor enzymatic activity or groups of enzymes or enzymatic activities froma complex mixture of materials, examples including, but not limited to,disrupted cell suspensions or cultured growth media. Thus a purifiedenzyme or protein should be understood to be an enzyme or protein thathas been separated or enriched from a complex matrix, wherein itsrelative concentration, as compared to other matrix components, isincreased. Methods for purifying an enzyme include, but are not limitedto, mechanical, chromatographic, chemical, pH or temperature means. Forexample, the addition of a salt to a disrupted cell suspension resultingin the precipitation of the target enzyme or protein followed bymechanical separation of the precipitated enzyme or protein, e.g.,membrane filtration or centrifugation. Further examples may include theseparation of an enzyme from a complex matrix through affinity basedchromatographic methods (e.g. hexa-histidine-tag or streptavidin basedpurification).

Enzymatic Specificity

Enzymatic specificity should be understood to be a trait inherent to anenzyme wherein it demonstrates improved reaction enzyme kinetics,thermodynamics or rates for one substrate as compared to anothersubstrate. Enzymes with high specificity are best exemplified by havinga high ratio of catalytic rate (defined as turnover number or Kcat) tothe Michaelis constant (Km) or Kcat/Km. It is advantageous to have anenzyme with high substrate specificity as this improves the rate of areaction and improves yield by decreasing the production of non-targetproducts. For example, the pathway described herein for the productionof allulose has several intermediates that are similar in chemicalstructure, namely gluclose 1-phosphate, glucose 6-phosphate, fructose6-phosphate and allulose 6-phosphate. The ultimate enzymatic step inthis process is the dephosphorylation of allulose 6-phosphate to theproduct allulose via an allulose 6-phosphate phosphatase. It isadvantageous to utilize an enzyme with a very high-specificity forallulose 6-phosphate and a relatively low specificity for the otherpathway intermediates, namely gluclose 1-phosphate glucose 6-phosphateand fructose 6-phosphate. Catalytic dephosphorylation of theseintermediates would result in the production of either glucose orfructose thus decreasing yield and increasing product complexity.

Additional Embodiments

1. A cell-free method for producing allulose, the method comprising:

(a) culturing cells engineered to express a thermostable α-glucanphosphorylase (also referred to as a starch phosphorylase), athermostable phosphoglucomutase, a thermostable phosphoglucoisomerase, athermostable allulose 6-phosphate epimerase, and a thermostable allulose6-phosphate phosphatase to produce cultured cells that express thethermostable enzymes;(b) lysing the cultured cells to produce a cell lysate;(c) heating the cell lysate to a temperature that inactivates nativeenzymatic activity but does not inactivate the thermostable enzymes ofstep (a) to produce a heat-inactivated lysate; and(d) incubating the heat-inactivated lysate in the presence of a starchand inorganic phosphate to produce allulose.2. A cell-free method for producing allulose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable α-glucan phosphorylases,thermostable phosphoglucomutases, thermostable phosphoglucoisomerases,thermostable allulose 6-phosphate epimerases, and thermostable allulose6-phosphate phosphatases to produce at least two cultured populations ofcells expressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture that comprises a thermostable α-glucan phosphorylase, athermostable phosphoglucomutase, a thermostable phosphoglucoisomerase, athermostable allulose 6-phosphate epimerase, and a thermostable allulose6-phosphate phosphatase;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (c) to produce a heat-inactivated lysate; and(e) incubating the reaction mixture in the presence of a starch andinorganic phosphate to produce allulose.3. A cell-free method for producing allulose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable α-glucan phosphorylases,thermostable phosphoglucomutases, thermostable phosphoglucoisomerases,thermostable allulose 6-phosphate epimerases, and thermostable allulose6-phosphate phosphatases to produce at least two cultured populations ofcells expressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (a) to produce a heat-inactivated lysate;(e) adding to the heat-inactivated lysate at least one purified enzymeselected from the group consisting of thermostable α-glucanphosphorylases, thermostable phosphoglucomutases, thermostablephosphoglucoisomerases, thermostable allulose 6-phosphate epimerases,and thermostable allulose 6-phosphate phosphatases to produce a reactionmixture comprising a α-glucan phosphorylase, a phosphoglucomutase, aphosphoglucoisomerase, an allulose 6-phosphate epimerase, and anallulose 6-phosphate phosphatase; and(f) incubating the reaction mixture in the presence of a starch andinorganic phosphate to produce allulose.4. The cell-free method of any one of embodiments 1-3, wherein thethermostable α-glucan phosphorylase(s) is selected from the groupconsisting of Aquifex aeolicus, Thermocrinis minervae,Thermosulfidibacter takaii, Thermosulfurimonas dismutans, Thermococcuslitoralis, Palaeococcus pacificus, Thermotoga neapolitana,Ruminiclostridium thermocellum, Pyrococcus abyssi, Thermococcusthioreducens, Deinococcus radiodurans, Sulfolobus acidocaldarius,Thermus caldophilus, Meiothermus silvanus, Oceanithermus profundus,Ardenticatena maritima, Thermococcus barophilus, Pseudothermotogathermarum, Hydrogenobacter thermophilus, Thermus oshimai, Meiothermusruber, and Marinitoga piezophila α-glucan phosphorylases.5. The cell-free method of any one of embodiments 1-4, wherein thethermostable phosphoglucomutase(s) is selected from the group consistingof Thermococcus kodakaraensis, Pyrococcus kukulkanii, Ammonifexdegensii, Methanothermobacter wolfeii, Methanothermus fervidus,Sulfolobus acidocaldarius, Archaeoglobus fulgidus, Ferroglobus placidus,Geoglobus ahangari, Archaeoglobus veneficus, Archaeoglobussulfaticallidus, Aciduliprofundum boonie, Clostridium thermocellum,Defluviitalea phaphyphila, Caminicella sporogenes, Caloranaerobacterferrireducens, Thermosipho malanesiensis, Fervidobacterium pennivorans,Symbiobacterium thermophilum, Spirochaeta thermophila, andThermoanaerobacter wiegelii phosphoglucomutases.6. The cell-free method of any one of embodiments 1-5, wherein thethermostable phosphoglucomutase(s) is selected from the group consistingof Thermus thermophilus, Meiothermus timidus, Thermus filiformis,Marinithermus hydrothermalis, Thermosipho africanus,Sulfurihydrogenibium azorense, Persephonella marina, Marinitogapiezophila, Kosmotoga olearia, Thermotoga maritima, Geobacillusstearothermophilus, Anoxybacillus flavithermus, Thermosulfidibactertakaii, Fervidobacterium nodosum, Clostridium thermocellum,Thermoanaerobacterium thermosaccharolyticum, Methanococcus jannaschii,Methanotorris igneus, Methanocaldococcus villosus, Methanothermococcusokinawensis, Pseudothermotoga thermarum, Deferribacter desulfuricans,and Thermovibrio ammonificans, phosphoglucomutases.7. The cell-free method of any one of embodiments 1-6, wherein thethermostable allulose 6-phosphate epimerase(s) is selected from thegroup consisting of Thermobacterium thermosaccharolyticum,Thermoanaerobacter brockii, Caldanaerobacter subterraneus, Deferribacterdesulfuricans, Thermocrinis ruber, Hydrogenivirga sp. 128-5-R1-1,Brevibacillus thermoruber, Thermosipho atlanticus, andThermosulfidibacter takaii allulose 6-phosphate epimerases.8. The cell-free method of any one of embodiments 1-7, wherein thethermostable allulose 6-phosphate phosphatase(s) is selected from thegroup consisting of Thermoanaerobacter wiegelii, Thermoanaerobacterethanolicus, Thermus islandicus, Deinococcus geothermalis DSM 11300,Thermosphaera aggregans, Crenarchaeota archaeon, Pyrococcus horikoshiiOt3, Aquifex aeolicus, Ruminiclostridium thermocellum, Desulfotomaculumkuznetsovii, Caldanaerobacter subterraneus, Acidothermus cellulolyticus,Methanothermobacter thermautotrophicus, Thermobifida fusca, Thermotoganeapolitana, Petrotoga mobilis, and Thermodesulfatator indicus, andThermus thermophilus allulose 6-phosphate phosphatases.9. A cell-free method for producing glucose, the method comprising:(a) culturing cells engineered to express a thermostable α-glucanphosphorylase, a thermostable phosphoglucomutase, and a thermostableglucose 6-phosphate phosphatase to produce cultured cells that expressthe thermostable enzymes;(b) lysing the cultured cells to produce a cell lysate;(c) heating the cell lysate to a temperature that inactivates nativeenzymatic activity but does not inactivate the thermostable enzymes ofstep (a) to produce a heat-inactivated lysate; and(d) incubating the heat-inactivated lysate in the presence of a starchand inorganic phosphate to produce glucose.10. A cell-free method for producing glucose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable α-glucan phosphorylases,thermostable phosphoglucomutases, and thermostable glucose 6-phosphatephosphatases to produce at least two cultured populations of cellsexpressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture that comprises a thermostable α-glucan phosphorylase, athermostable phosphoglucomutase, and a thermostable glucose 6-phosphatephosphatase;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (c) to produce a heat-inactivated lysate; and(e) incubating the reaction mixture in the presence of a starch andinorganic phosphate to produce glucose.11. A cell-free method for producing glucose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable α-glucan phosphorylases,thermostable phosphoglucomutases, and thermostable glucose 6-phosphatephosphatases to produce at least two cultured populations of cellsexpressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (a) to produce a heat-inactivated lysate;(e) adding to the heat-inactivated lysate at least one purified enzymeselected from the group consisting of thermostable α-glucanphosphorylases, thermostable phosphoglucomutases, and thermostableglucose 6-phosphate phosphatases to produce a reaction mixturecomprising a α-glucan phosphorylase, a phosphoglucomutase, and a glucose6-phosphate phosphatase; and(f) incubating the reaction mixture in the presence of a starch andinorganic phosphate to produce glucose.12. A cell-free method for producing fructose, the method comprising:(a) culturing cells engineered to express a thermostable α-glucanphosphorylase, a thermostable phosphoglucomutase, a thermostablephosphoglucoisomerase, and a thermostable fructose 6-phosphatephosphatase to produce cultured cells that express the thermostableenzymes;(b) lysing the cultured cells to produce a cell lysate;(c) heating the cell lysate to a temperature that inactivates nativeenzymatic activity but does not inactivate the thermostable enzymes ofstep (a) to produce a heat-inactivated lysate; and(d) incubating the heat-inactivated lysate in the presence of a starchand inorganic phosphate to produce fructose.13. A cell-free method for producing fructose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable α-glucan phosphorylases,thermostable phosphoglucomutases, thermostable phosphoglucoisomerases,and thermostable fructose 6-phosphate phosphatases to produce at leasttwo cultured populations of cells expressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture that comprises a thermostable α-glucan phosphorylase, athermostable phosphoglucomutase, a thermostable phosphoglucoisomerase,and a thermostable fructose 6-phosphate phosphatase;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (c) to produce a heat-inactivated lysate; and(e) incubating the reaction mixture in the presence of a starch andinorganic phosphate to produce fructose.14. A cell-free method for producing fructose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable α-glucan phosphorylases,thermostable phosphoglucomutases, thermostable phosphoglucoisomerases,and thermostable fructose 6-phosphate phosphatases to produce at leasttwo cultured populations of cells expressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (a) to produce a heat-inactivated lysate;(e) adding to the heat-inactivated lysate at least one purified enzymeselected from the group consisting of thermostable α-glucanphosphorylases, thermostable phosphoglucomutases, thermostablephosphoglucoisomerases, and thermostable fructose 6-phosphatephosphatases to produce a reaction mixture comprising a α-glucanphosphorylase, a phosphoglucomutase, a phosphoglucoisomerase, and afructose 6-phosphate phosphatase; and(f) incubating the reaction mixture in the presence of a starch andinorganic phosphate to produce fructose.15. A cell-free method for producing sorbitol, the method comprising:(a) culturing cells engineered to express a thermostable α-glucanphosphorylase, a thermostable phosphoglucomutase, a thermostable aldosedehydrogenase, and a thermostable sorbitol-6-phosphate phosphatase toproduce cultured cells that express the thermostable enzymes;(b) lysing the cultured cells to produce a cell lysate;(c) heating the cell lysate to a temperature that inactivates nativeenzymatic activity but does not inactivate the thermostable enzymes ofstep (a) to produce a heat-inactivated lysate; and(d) incubating the heat-inactivated lysate in the presence of a starchand inorganic phosphate to produce sorbitol.16. A cell-free method for producing sorbitol, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable α-glucan phosphorylases,thermostable phosphoglucomutases, thermostable aldose dehydrogenases,and thermostable sorbitol-6-phosphate phosphatases to produce at leasttwo cultured populations of cells expressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture that comprises a thermostable α-glucan phosphorylase, athermostable phosphoglucomutase, a thermostable aldose dehydrogenase,and a thermostable sorbitol-6-phosphate phosphatase;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (c) to produce a heat-inactivated lysate; and(e) incubating the reaction mixture in the presence of a starch andinorganic phosphate to produce sorbitol.17. A cell-free method for producing sorbitol, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable α-glucan phosphorylases,thermostable phosphoglucomutases, thermostable aldose dehydrogenases,and thermostable sorbitol-6-phosphate phosphatases to produce at leasttwo cultured populations of cells expressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (a) to produce a heat-inactivated lysate;(e) adding to the heat-inactivated lysate at least one purified enzymeselected from the group consisting of thermostable α-glucanphosphorylases, thermostable phosphoglucomutases, thermostable aldosedehydrogenases, and thermostable sorbitol-6-phosphate phosphatases toproduce a reaction mixture comprising a α-glucan phosphorylase, aphosphoglucomutase, an aldose dehydrogenase, and a sorbitol-6-phosphatephosphatase; and(f) incubating the reaction mixture in the presence of a starch andinorganic phosphate to produce sorbitol.18. A cell-free method for producing ribulose, the method comprising:(a) culturing cells engineered to express a thermostable α-glucanphosphorylase, a thermostable phosphoglucomutase, a thermostable glucose6-phosphate dehydrogenase, a thermostable 6-phosphogluconolactonase, athermostable 6-phosphogluconate dehydrogenase, and a thermostableribulose 5-phosphate phosphatase to produce cultured cells that expressthe thermostable enzymes;(b) lysing the cultured cells to produce a cell lysate;(c) heating the cell lysate to a temperature that inactivates nativeenzymatic activity but does not inactivate the thermostable enzymes ofstep (a) to produce a heat-inactivated lysate; and(d) incubating the heat-inactivated lysate in the presence of a starchand inorganic phosphate to produce ribulose.19. A cell-free method for producing ribulose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable α-glucan phosphorylases,thermostable phosphoglucomutases, thermostable glucose 6-phosphatedehydrogenases, thermostable 6-phosphogluconolactonases, thermostable6-phosphogluconate dehydrogenases, and thermostable ribulose 5-phosphatephosphatases to produce at least two cultured populations of cellsexpressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture that comprises a thermostable α-glucan phosphorylase, athermostable phosphoglucomutase, a thermostable glucose 6-phosphatedehydrogenase, a thermostable 6-phosphogluconolactonase, a thermostable6-phosphogluconate dehydrogenase, and a thermostable ribulose5-phosphate phosphatase;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (c) to produce a heat-inactivated lysate; and(e) incubating the reaction mixture in the presence of a starch andinorganic phosphate to produce ribulose.20. A cell-free method for producing ribulose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable α-glucan phosphorylases,thermostable phosphoglucomutases, thermostable glucose 6-phosphatedehydrogenases, thermostable 6-phosphogluconolactonases, thermostable6-phosphogluconate dehydrogenases, and thermostable ribulose 5-phosphatephosphatases to produce at least two cultured populations of cellsexpressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (a) to produce a heat-inactivated lysate;(e) adding to the heat-inactivated lysate at least one purified enzymeselected from the group consisting of thermostable α-glucanphosphorylases, thermostable phosphoglucomutases, thermostable glucose6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases,thermostable 6-phosphogluconate dehydrogenases, and thermostableribulose 5-phosphate phosphatases to produce a reaction mixturecomprising a α-glucan phosphorylase, a phosphoglucomutase, a glucose6-phosphate dehydrogenase, a 6-phosphogluconolactonase, a6-phosphogluconate dehydrogenase, and a ribulose 5-phosphatephosphatase; and(f) incubating the reaction mixture in the presence of a starch andinorganic phosphate to produce ribulose.21. A cell-free method for producing ribose, the method comprising:(a) culturing cells engineered to express a thermostable α-glucanphosphorylase, a thermostable phosphoglucomutase, a thermostable glucose6-phosphate dehydrogenase, a thermostable 6-phosphogluconolactonase, athermostable 6-phosphogluconate dehydrogenase, a thermostable ribose5-phosphate isomerase, and a thermostable ribose 5-phosphate phosphataseto produce cultured cells that express the thermostable enzymes;(b) lysing the cultured cells to produce a cell lysate;(c) heating the cell lysate to a temperature that inactivates nativeenzymatic activity but does not inactivate the thermostable enzymes ofstep (a) to produce a heat-inactivated lysate; and(d) incubating the heat-inactivated lysate in the presence of a starchand inorganic phosphate to produce ribose.22. A cell-free method for producing ribose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable α-glucan phosphorylases,thermostable phosphoglucomutases, thermostable glucose 6-phosphatedehydrogenases, thermostable 6-phosphogluconolactonases, thermostable6-phosphogluconate dehydrogenases, thermostable ribose 5-phosphateisomerases, and thermostable ribose 5-phosphate phosphatases to produceat least two cultured populations of cells expressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture that comprises a thermostable α-glucan phosphorylase, athermostable phosphoglucomutase, a thermostable glucose 6-phosphatedehydrogenase, a thermostable 6-phosphogluconolactonase, a thermostable6-phosphogluconate dehydrogenase, a thermostable ribose 5-phosphateisomerase, and a thermostable ribose 5-phosphate phosphatase;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (c) to produce a heat-inactivated lysate; and(e) incubating the reaction mixture in the presence of a starch andinorganic phosphate to produce ribose.23. A cell-free method for producing ribose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable α-glucan phosphorylases,thermostable phosphoglucomutases, thermostable glucose 6-phosphatedehydrogenases, thermostable 6-phosphogluconolactonases, thermostable6-phosphogluconate dehydrogenases, thermostable ribose 5-phosphateisomerases, and thermostable ribose 5-phosphate phosphatases to produceat least two cultured populations of cells expressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (a) to produce a heat-inactivated lysate;(e) adding to the heat-inactivated lysate at least one purified enzymeselected from the group consisting of thermostable α-glucanphosphorylases, thermostable phosphoglucomutases, thermostable glucose6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases,thermostable 6-phosphogluconate dehydrogenases, thermostable ribose5-phosphate isomerases, and thermostable ribose 5-phosphate phosphatasesto produce a reaction mixture comprising a α-glucan phosphorylase, aphosphoglucomutase, a glucose 6-phosphate dehydrogenase, a6-phosphogluconolactonase, a 6-phosphogluconate dehydrogenase, a ribose5-phosphate isomerase, and a ribose 5-phosphate phosphatase; and(f) incubating the reaction mixture in the presence of a starch andinorganic phosphate to produce ribose.24. A cell-free method for producing arabinose, the method comprising:(a) culturing cells engineered to express a thermostable α-glucanphosphorylase, a thermostable phosphoglucomutase, a thermostable glucose6-phosphate dehydrogenase, a thermostable 6-phosphogluconolactonase, athermostable 6-phosphogluconate dehydrogenase, a thermostable arabinose5-phosphate isomerase, and a thermostable arabinose 5-phosphatephosphatase to produce cultured cells that express the thermostableenzymes;(b) lysing the cultured cells to produce a cell lysate;(c) heating the cell lysate to a temperature that inactivates nativeenzymatic activity but does not inactivate the thermostable enzymes ofstep (a) to produce a heat-inactivated lysate; and(d) incubating the heat-inactivated lysate in the presence of a starchand inorganic phosphate to produce arabinose.25. A cell-free method for producing arabinose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable α-glucan phosphorylases,thermostable phosphoglucomutases, thermostable glucose 6-phosphatedehydrogenases, thermostable 6-phosphogluconolactonases, thermostable6-phosphogluconate dehydrogenases, thermostable arabinose 5-phosphateisomerases, and thermostable arabinose 5-phosphate phosphatases toproduce at least two cultured populations of cells expressing differentenzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture that comprises a thermostable α-glucan phosphorylase, athermostable phosphoglucomutase, a thermostable glucose 6-phosphatedehydrogenase, a thermostable 6-phosphogluconolactonase, a thermostable6-phosphogluconate dehydrogenase, a thermostable arabinose 5-phosphateisomerase, and a thermostable arabinose 5-phosphate phosphatase;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (c) to produce a heat-inactivated lysate; and(e) incubating the reaction mixture in the presence of a starch andinorganic phosphate to produce arabinose.26. A cell-free method for producing arabinose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable α-glucan phosphorylases,thermostable phosphoglucomutases, thermostable glucose 6-phosphatedehydrogenases, thermostable 6-phosphogluconolactonases, thermostable6-phosphogluconate dehydrogenases, thermostable arabinose 5-phosphateisomerases, and thermostable arabinose 5-phosphate phosphatases toproduce at least two cultured populations of cells expressing differentenzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (a) to produce a heat-inactivated lysate;(e) adding to the heat-inactivated lysate at least one purified enzymeselected from the group consisting of thermostable α-glucanphosphorylases, thermostable phosphoglucomutases, thermostable glucose6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases,thermostable 6-phosphogluconate dehydrogenases, thermostable arabinose5-phosphate isomerases, and thermostable arabinose 5-phosphatephosphatases to produce a reaction mixture comprising a α-glucanphosphorylase, a phosphoglucomutase, a glucose 6-phosphatedehydrogenase, a 6-phosphogluconolactonase, a 6-phosphogluconatedehydrogenase, an arabinose 5-phosphate isomerase, and an arabinose5-phosphate phosphatase; and(f) incubating the reaction mixture in the presence of a starch andinorganic phosphate to produce arabinose.27. A cell-free method for producing mannose, the method comprising:(a) culturing cells engineered to express a thermostable α-glucanphosphorylase, a thermostable phosphoglucomutase, a thermostablephosphoglucoisomerase, a thermostable mannose 6-phosphate epimerase, anda thermostable mannose 6-phosphate phosphatase to produce cultured cellsthat express the thermostable enzymes;(b) lysing the cultured cells to produce a cell lysate;(c) heating the cell lysate to a temperature that inactivates nativeenzymatic activity but does not inactivate the thermostable enzymes ofstep (a) to produce a heat-inactivated lysate; and(d) incubating the heat-inactivated lysate in the presence of a starchand inorganic phosphate to produce mannose.28. A cell-free method for producing mannose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable α-glucan phosphorylases,thermostable phosphoglucomutases, thermostable phosphoglucoisomerases,thermostable mannose 6-phosphate epimerases, and thermostable mannose6-phosphate phosphatases to produce at least two cultured populations ofcells expressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture that comprises a thermostable α-glucan phosphorylase, athermostable phosphoglucomutase, a thermostable phosphoglucoisomerase, athermostable mannose 6-phosphate epimerase, and a thermostable mannose6-phosphate phosphatase;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (c) to produce a heat-inactivated lysate; and(e) incubating the reaction mixture in the presence of a starch andinorganic phosphate to produce mannose.29. A cell-free method for producing mannose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable α-glucan phosphorylases,thermostable phosphoglucomutases, thermostable phosphoglucoisomerases,thermostable mannose 6-phosphate epimerases, and thermostable mannose6-phosphate phosphatases to produce at least two cultured populations ofcells expressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (a) to produce a heat-inactivated lysate;(e) adding to the heat-inactivated lysate at least one purified enzymeselected from the group consisting of thermostable α-glucanphosphorylases, thermostable phosphoglucomutases, thermostablephosphoglucoisomerases, thermostable mannose 6-phosphate epimerases, andthermostable mannose 6-phosphate phosphatases to produce a reactionmixture comprising a α-glucan phosphorylase, a phosphoglucomutase, aphosphoglucoisomerase, an mannose 6-phosphate epimerase, and an mannose6-phosphate phosphatase; and(f) incubating the reaction mixture in the presence of a starch andinorganic phosphate to produce mannose.30. The method of any one of embodiments 1-26, wherein the cellscomprise bacterial cells.31. The method of any one of embodiments 1-26, wherein the cellscomprise yeast cells.32. The method for any one of embodiments 1-31, wherein at least one ofthe enzymes is heterologous to the cells.33. The method of any one of embodiments 1-32, wherein lysing step (b)comprises mechanically, chemically, or enzymatically lysing the culturedcells.34. The method for any one of embodiments 1-33, wherein heating step (c)comprises heating the cell lysate to a temperature of at least 50° C.35. The method for any one of embodiments 1-34, wherein the starchcomprises amylose, amylopectin, or both amylose and amylopectin.36. The method for any one of embodiments 1-35, wherein the thermostableα-glucan phosphorylase and the thermostable phosphoglucomutase areexpressed as a single fusion protein or a bifunctional protein.37. A cell lysate produced by the method for any one of embodiments1-36.38. An engineered cell comprising a α-glucan phosphorylase, aphosphoglucomutase, and a glucose 6-phosphate phosphatase, optionallywherein at least one of the foregoing enzymes is a thermostable enzyme.39. An engineered cell comprising a α-glucan phosphorylase, aphosphoglucomutase, a phosphoglucoisomerase, and a fructose 6-phosphatephosphatase, optionally wherein at least one of the foregoing enzymes isa thermostable enzyme.40. An engineered cell comprising a α-glucan phosphorylase, aphosphoglucomutase, a phosphoglucoisomerase, an allulose 6-phosphateepimerase, and an allulose 6-phosphate phosphatase, optionally whereinat least one of the foregoing enzymes is a thermostable enzyme.41. An engineered cell comprising a α-glucan phosphorylase, aphosphoglucomutase, a aldose dehydrogenase, and a sorbitol-6-phosphatephosphatase, optionally wherein at least one of the foregoing enzymes isa thermostable enzyme.42. An engineered cell comprising a α-glucan phosphorylase, aphosphoglucomutase, a glucose 6-phosphate dehydrogenase, a6-phosphogluconolactonase, a 6-phosphogluconate dehydrogenase, and aribulose 5-phosphate phosphatase, optionally wherein at least one of theforegoing enzymes is a thermostable enzyme.43. An engineered cell comprising a α-glucan phosphorylase, aphosphoglucomutase, a glucose 6-phosphate dehydrogenase, a6-phosphogluconolactonase, a 6-phosphogluconate dehydrogenase, a ribose5-phosphate isomerase, and a ribose 5-phosphate phosphatase, optionallywherein at least one of the foregoing enzymes is a thermostable enzyme.44. An engineered cell comprising a α-glucan phosphorylase, aphosphoglucomutase, a glucose 6-phosphate dehydrogenase, a6-phosphogluconolactonase, a 6-phosphogluconate dehydrogenase, anarabinose 5-phosphate isomerase, and an arabinose 5-phosphatephosphatase, optionally wherein at least one of the foregoing enzymes isa thermostable enzyme.45. An engineered cell comprising a α-glucan phosphorylase, aphosphoglucomutase, a phosphoglucoisomerase, a mannose 6-phosphateepimerase, and a mannose 6-phosphate phosphatase, optionally wherein atleast one of the foregoing enzymes is a thermostable enzyme.46. The engineered cell of any one of embodiments embodiment 38-45,wherein the cell is a bacterial cell or a yeast cell.47. A single cell lysate, a mixture of cell lysates obtained from atleast two cell populations, or a reaction mixture comprising a α-glucanphosphorylase, a phosphoglucomutase, and a glucose 6-phosphatephosphatase, optionally wherein at least one of the foregoing enzymes isa thermostable enzyme.48. A single cell lysate, a mixture of cell lysates obtained from atleast two cell populations, or a reaction mixture comprising a α-glucanphosphorylase, a phosphoglucomutase, a phosphoglucoisomerase, and afructose 6-phosphate phosphatase, optionally wherein at least one of theforegoing enzymes is a thermostable enzyme.49. A single cell lysate, a mixture of cell lysates obtained from atleast two cell populations, or a reaction mixture comprising a α-glucanphosphorylase, a phosphoglucomutase, a phosphoglucoisomerase, anallulose 6-phosphate epimerase, and an allulose 6-phosphate phosphatase,optionally wherein at least one of the foregoing enzymes is athermostable enzyme.50. A single cell lysate, a mixture of cell lysates obtained from atleast two cell populations, or a reaction mixture comprising a α-glucanphosphorylase, a phosphoglucomutase, a aldose dehydrogenase, and asorbitol-6-phosphate phosphatase, optionally wherein at least one of theforegoing enzymes is a thermostable enzyme.51. A single cell lysate, a mixture of cell lysates obtained from atleast two cell populations, or a reaction mixture comprising a α-glucanphosphorylase, a phosphoglucomutase, a glucose 6-phosphatedehydrogenase, a 6-phosphogluconolactonase, a 6-phosphogluconatedehydrogenase, and a ribulose 5-phosphate phosphatase, optionallywherein at least one of the foregoing enzymes is a thermostable enzyme.52. A single cell lysate, a mixture of cell lysates obtained from atleast two cell populations, or a reaction mixture comprising a α-glucanphosphorylase, a phosphoglucomutase, a glucose 6-phosphatedehydrogenase, a 6-phosphogluconolactonase, a 6-phosphogluconatedehydrogenase, a ribose 5-phosphate isomerase, and a ribose 5-phosphatephosphatase, optionally wherein at least one of the foregoing enzymes isa thermostable enzyme.53. A single cell lysate, a mixture of cell lysates obtained from atleast two cell populations, or a reaction mixture comprising a α-glucanphosphorylase, a phosphoglucomutase, a glucose 6-phosphatedehydrogenase, a 6-phosphogluconolactonase, a 6-phosphogluconatedehydrogenase, an arabinose 5-phosphate isomerase, and an arabinose5-phosphate phosphatase, optionally wherein at least one of theforegoing enzymes is a thermostable enzyme.54. A single cell lysate, a mixture of cell lysates obtained from atleast two cell populations, or a reaction mixture comprising a α-glucanphosphorylase, a phosphoglucomutase, a phosphoglucoisomerase, an mannose6-phosphate epimerase, and an mannose 6-phosphate phosphatase,optionally wherein at least one of the foregoing enzymes is athermostable enzyme.55. A cell-free method for producing allulose, the method comprising:(a) culturing cells engineered to express a thermostable cellodextrinphosphorylase, a thermostable phosphoglucomutase, a thermostablephosphoglucoisomerase, a thermostable allulose 6-phosphate epimerase,and a thermostable allulose 6-phosphate phosphatase to produce culturedcells that express the thermostable enzymes;(b) lysing the cultured cells to produce a cell lysate;(c) heating the cell lysate to a temperature that inactivates nativeenzymatic activity but does not inactivate the thermostable enzymes ofstep (a) to produce a heat-inactivated lysate; and(d) incubating the heat-inactivated lysate in the presence of acellodextrin and inorganic phosphate to produce allulose.56. A cell-free method for producing allulose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable cellodextrin phosphorylases,thermostable phosphoglucomutases, thermostable phosphoglucoisomerases,thermostable allulose 6-phosphate epimerases, and thermostable allulose6-phosphate phosphatases to produce at least two cultured populations ofcells expressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture that comprises a thermostable cellodextrin phosphorylase, athermostable phosphoglucomutase, a thermostable phosphoglucoisomerase, athermostable allulose 6-phosphate epimerase, and a thermostable allulose6-phosphate phosphatase;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (c) to produce a heat-inactivated lysate; and(e) incubating the reaction mixture in the presence of a cellodextrinand inorganic phosphate to produce allulose.57. A cell-free method for producing allulose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable cellodextrin phosphorylases,thermostable phosphoglucomutases, thermostable phosphoglucoisomerases,thermostable allulose 6-phosphate epimerases, and thermostable allulose6-phosphate phosphatases to produce at least two cultured populations ofcells expressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (a) to produce a heat-inactivated lysate;(e) adding to the heat-inactivated lysate at least one purified enzymeselected from the group consisting of thermostable cellodextrinphosphorylases, thermostable phosphoglucomutases, thermostablephosphoglucoisomerases, thermostable allulose 6-phosphate epimerases,and thermostable allulose 6-phosphate phosphatases to produce a reactionmixture comprising a cellodextrin phosphorylase, a phosphoglucomutase, aphosphoglucoisomerase, an allulose 6-phosphate epimerase, and anallulose 6-phosphate phosphatase; and(f) incubating the reaction mixture in the presence of a cellodextrinand inorganic phosphate to produce allulose.58. The cell-free method of any one of embodiments 55-57, wherein thethermostable cellodextrin phosphorylase(s) is selected from the groupconsisting of Aquifex aeolicus, Thermocrinis minervae,Thermosulfidibacter takaii, Thermosulfurimonas dismutans, Thermococcuslitoralis, Palaeococcus pacificus, Thermotoga neapolitana,Ruminiclostridium thermocellum, Pyrococcus abyssi, Thermococcusthioreducens, Deinococcus radiodurans, Sulfolobus acidocaldarius,Thermus caldophilus, Meiothermus silvanus, Oceanithermus profundus,Ardenticatena maritima, Thermococcus barophilus, Pseudothermotogathermarum, Hydrogenobacter thermophilus, Thermus oshimai, Meiothermusruber, and Marinitoga piezophila cellodextrin phosphorylases.59. The cell-free method of any one of embodiments 55-58, wherein thethermostable phosphoglucomutase(s) is selected from the group consistingof Thermococcus kodakaraensis, Pyrococcus kukulkanii, Ammonifexdegensii, Methanothermobacter wolfeii, Methanothermus fervidus,Sulfolobus acidocaldarius, Archaeoglobus fulgidus, Ferroglobus placidus,Geoglobus ahangari, Archaeoglobus veneficus, Archaeoglobussulfaticallidus, Aciduliprofundum boonie, Clostridium thermocellum,Defluviitalea phaphyphila, Caminicella sporogenes, Caloranaerobacterferrireducens, Thermosipho malanesiensis, Fervidobacterium pennivorans,Symbiobacterium thermophilum, Spirochaeta thermophila, andThermoanaerobacter wiegelii phosphoglucomutases.60. The cell-free method of any one of embodiments 55-59, wherein thethermostable phosphoglucomutase(s) is selected from the group consistingof Thermus thermophilus, Meiothermus timidus, Thermus filiformis,Marinithermus hydrothermalis, Thermosipho africanus,Sulfurihydrogenibium azorense, Persephonella marina, Marinitogapiezophila, Kosmotoga olearia, Thermotoga maritima, Geobacillusstearothermophilus, Anoxybacillus flavithermus, Thermosulfidibactertakaii, Fervidobacterium nodosum, Clostridium thermocellum,Thermoanaerobacterium thermosaccharolyticum, Methanococcus jannaschii,Methanotorris igneus, Methanocaldococcus villosus, Methanothermococcusokinawensis, Pseudothermotoga thermarum, Deferribacter desulfuricans,and Thermovibrio ammonificans, phosphoglucomutases.61. The cell-free method of any one of embodiments 55-60, wherein thethermostable allulose 6-phosphate epimerase(s) is selected from thegroup consisting of Thermobacterium thermosaccharolyticum,Thermoanaerobacter brockii, Caldanaerobacter subterraneus, Deferribacterdesulfuricans, Thermocrinis ruber, Hydrogenivirga sp. 128-5-R1-1,Brevibacillus thermoruber, Thermosipho atlanticus, andThermosulfidibacter takaii allulose 6-phosphate epimerases.62. The cell-free method of any one of embodiments 55-61, wherein thethermostable allulose 6-phosphate phosphatase(s) is selected from thegroup consisting of Thermoanaerobacter wiegelii, Thermoanaerobacterethanolicus, Thermus islandicus, Deinococcus geothermalis DSM 11300,Thermosphaera aggregans, Crenarchaeota archaeon, Pyrococcus horikoshiiOt3, Aquifex aeolicus, Ruminiclostridium thermocellum, Desulfotomaculumkuznetsovii, Caldanaerobacter subterraneus, Acidothermus cellulolyticus,Methanothermobacter thermautotrophicus, Thermobifida fusca, Thermotoganeapolitana, Petrotoga mobilis, and Thermodesulfatator indicus, andThermus thermophilus allulose 6-phosphate phosphatases.63. A cell-free method for producing glucose, the method comprising:(a) culturing cells engineered to express a thermostable cellodextrinphosphorylase, a thermostable phosphoglucomutase, and a thermostableglucose 6-phosphate phosphatase to produce cultured cells that expressthe thermostable enzymes;(b) lysing the cultured cells to produce a cell lysate;(c) heating the cell lysate to a temperature that inactivates nativeenzymatic activity but does not inactivate the thermostable enzymes ofstep (a) to produce a heat-inactivated lysate; and(d) incubating the heat-inactivated lysate in the presence of acellodextrin and inorganic phosphate to produce glucose.64. A cell-free method for producing glucose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable cellodextrin phosphorylases,thermostable phosphoglucomutases, and thermostable glucose 6-phosphatephosphatases to produce at least two cultured populations of cellsexpressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture that comprises a thermostable cellodextrin phosphorylase, athermostable phosphoglucomutase, and a thermostable glucose 6-phosphatephosphatase;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (c) to produce a heat-inactivated lysate; and(e) incubating the reaction mixture in the presence of a cellodextrinand inorganic phosphate to produce glucose.65. A cell-free method for producing glucose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable cellodextrin phosphorylases,thermostable phosphoglucomutases, and thermostable glucose 6-phosphatephosphatases to produce at least two cultured populations of cellsexpressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (a) to produce a heat-inactivated lysate;(e) adding to the heat-inactivated lysate at least one purified enzymeselected from the group consisting of thermostable cellodextrinphosphorylases, thermostable phosphoglucomutases, and thermostableglucose 6-phosphate phosphatases to produce a reaction mixturecomprising a cellodextrin phosphorylase, a phosphoglucomutase, and aglucose 6-phosphate phosphatase; and(f) incubating the reaction mixture in the presence of a cellodextrinand inorganic phosphate to produce glucose.66. A cell-free method for producing fructose, the method comprising:(a) culturing cells engineered to express a thermostable cellodextrinphosphorylase, a thermostable phosphoglucomutase, a thermostablephosphoglucoisomerase, and a thermostable fructose 6-phosphatephosphatase to produce cultured cells that express the thermostableenzymes;(b) lysing the cultured cells to produce a cell lysate;(c) heating the cell lysate to a temperature that inactivates nativeenzymatic activity but does not inactivate the thermostable enzymes ofstep (a) to produce a heat-inactivated lysate; and(d) incubating the heat-inactivated lysate in the presence of acellodextrin and inorganic phosphate to produce fructose.67. A cell-free method for producing fructose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable cellodextrin phosphorylases,thermostable phosphoglucomutases, thermostable phosphoglucoisomerases,and thermostable fructose 6-phosphate phosphatases to produce at leasttwo cultured populations of cells expressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture that comprises a thermostable cellodextrin phosphorylase, athermostable phosphoglucomutase, a thermostable phosphoglucoisomerase,and a thermostable fructose 6-phosphate phosphatase;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (c) to produce a heat-inactivated lysate; and(e) incubating the reaction mixture in the presence of a cellodextrinand inorganic phosphate to produce fructose.68. A cell-free method for producing fructose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable cellodextrin phosphorylases,thermostable phosphoglucomutases, thermostable phosphoglucoisomerases,and thermostable fructose 6-phosphate phosphatases to produce at leasttwo cultured populations of cells expressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (a) to produce a heat-inactivated lysate;(e) adding to the heat-inactivated lysate at least one purified enzymeselected from the group consisting of thermostable cellodextrinphosphorylases, thermostable phosphoglucomutases, thermostablephosphoglucoisomerases, and thermostable fructose 6-phosphatephosphatases to produce a reaction mixture comprising a cellodextrinphosphorylase, a phosphoglucomutase, a phosphoglucoisomerase, and afructose 6-phosphate phosphatase; and(f) incubating the reaction mixture in the presence of a cellodextrinand inorganic phosphate to produce fructose.69. A cell-free method for producing sorbitol, the method comprising:(a) culturing cells engineered to express a thermostable cellodextrinphosphorylase, a thermostable phosphoglucomutase, a thermostable aldosedehydrogenase, and a thermostable sorbitol-6-phosphate phosphatase toproduce cultured cells that express the thermostable enzymes;(b) lysing the cultured cells to produce a cell lysate;(c) heating the cell lysate to a temperature that inactivates nativeenzymatic activity but does not inactivate the thermostable enzymes ofstep (a) to produce a heat-inactivated lysate; and(d) incubating the heat-inactivated lysate in the presence of acellodextrin and inorganic phosphate to produce sorbitol.70. A cell-free method for producing sorbitol, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable cellodextrin phosphorylases,thermostable phosphoglucomutases, thermostable aldose dehydrogenases,and thermostable sorbitol-6-phosphate phosphatases to produce at leasttwo cultured populations of cells expressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture that comprises a thermostable cellodextrin phosphorylase, athermostable phosphoglucomutase, a thermostable aldose dehydrogenase,and a thermostable sorbitol-6-phosphate phosphatase;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (c) to produce a heat-inactivated lysate; and(e) incubating the reaction mixture in the presence of a cellodextrinand inorganic phosphate to produce sorbitol.71. A cell-free method for producing sorbitol, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable cellodextrin phosphorylases,thermostable phosphoglucomutases, thermostable aldose dehydrogenases,and thermostable sorbitol-6-phosphate phosphatases to produce at leasttwo cultured populations of cells expressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (a) to produce a heat-inactivated lysate;(e) adding to the heat-inactivated lysate at least one purified enzymeselected from the group consisting of thermostable cellodextrinphosphorylases, thermostable phosphoglucomutases, thermostable aldosedehydrogenases, and thermostable sorbitol-6-phosphate phosphatases toproduce a reaction mixture comprising a cellodextrin phosphorylase, aphosphoglucomutase, an aldose dehydrogenase, and a sorbitol-6-phosphatephosphatase; and(f) incubating the reaction mixture in the presence of a cellodextrinand inorganic phosphate to produce sorbitol.72. A cell-free method for producing ribulose, the method comprising:(a) culturing cells engineered to express a thermostable cellodextrinphosphorylase, a thermostable phosphoglucomutase, a thermostable glucose6-phosphate dehydrogenase, a thermostable 6-phosphogluconolactonase, athermostable 6-phosphogluconate dehydrogenase, and a thermostableribulose 5-phosphate phosphatase to produce cultured cells that expressthe thermostable enzymes;(b) lysing the cultured cells to produce a cell lysate;(c) heating the cell lysate to a temperature that inactivates nativeenzymatic activity but does not inactivate the thermostable enzymes ofstep (a) to produce a heat-inactivated lysate; and(d) incubating the heat-inactivated lysate in the presence of acellodextrin and inorganic phosphate to produce ribulose.73. A cell-free method for producing ribulose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable cellodextrin phosphorylases,thermostable phosphoglucomutases, thermostable glucose 6-phosphatedehydrogenases, thermostable 6-phosphogluconolactonases, thermostable6-phosphogluconate dehydrogenases, and thermostable ribulose 5-phosphatephosphatases to produce at least two cultured populations of cellsexpressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture that comprises a thermostable cellodextrin phosphorylase, athermostable phosphoglucomutase, a thermostable glucose 6-phosphatedehydrogenase, a thermostable 6-phosphogluconolactonase, a thermostable6-phosphogluconate dehydrogenase, and a thermostable ribulose5-phosphate phosphatase;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (c) to produce a heat-inactivated lysate; and(e) incubating the reaction mixture in the presence of a cellodextrinand inorganic phosphate to produce ribulose.74. A cell-free method for producing ribulose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable cellodextrin phosphorylases,thermostable phosphoglucomutases, thermostable glucose 6-phosphatedehydrogenases, thermostable 6-phosphogluconolactonases, thermostable6-phosphogluconate dehydrogenases, and thermostable ribulose 5-phosphatephosphatases to produce at least two cultured populations of cellsexpressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (a) to produce a heat-inactivated lysate;(e) adding to the heat-inactivated lysate at least one purified enzymeselected from the group consisting of thermostable cellodextrinphosphorylases, thermostable phosphoglucomutases, thermostable glucose6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases,thermostable 6-phosphogluconate dehydrogenases, and thermostableribulose 5-phosphate phosphatases to produce a reaction mixturecomprising a cellodextrin phosphorylase, a phosphoglucomutase, a glucose6-phosphate dehydrogenase, a 6-phosphogluconolactonase, a6-phosphogluconate dehydrogenase, and a ribulose 5-phosphatephosphatase; and(f) incubating the reaction mixture in the presence of a cellodextrinand inorganic phosphate to produce ribulose.75. A cell-free method for producing ribose, the method comprising:(a) culturing cells engineered to express a thermostable cellodextrinphosphorylase, a thermostable phosphoglucomutase, a thermostable glucose6-phosphate dehydrogenase, a thermostable 6-phosphogluconolactonase, athermostable 6-phosphogluconate dehydrogenase, a thermostable ribose5-phosphate isomerase, and a thermostable ribose 5-phosphate phosphataseto produce cultured cells that express the thermostable enzymes;(b) lysing the cultured cells to produce a cell lysate;(c) heating the cell lysate to a temperature that inactivates nativeenzymatic activity but does not inactivate the thermostable enzymes ofstep (a) to produce a heat-inactivated lysate; and(d) incubating the heat-inactivated lysate in the presence of acellodextrin and inorganic phosphate to produce ribose.76. A cell-free method for producing ribose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable cellodextrin phosphorylases,thermostable phosphoglucomutases, thermostable glucose 6-phosphatedehydrogenases, thermostable 6-phosphogluconolactonases, thermostable6-phosphogluconate dehydrogenases, thermostable ribose 5-phosphateisomerases, and thermostable ribose 5-phosphate phosphatases to produceat least two cultured populations of cells expressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture that comprises a thermostable cellodextrin phosphorylase, athermostable phosphoglucomutase, a thermostable glucose 6-phosphatedehydrogenase, a thermostable 6-phosphogluconolactonase, a thermostable6-phosphogluconate dehydrogenase, a thermostable ribose 5-phosphateisomerase, and a thermostable ribose 5-phosphate phosphatase;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (c) to produce a heat-inactivated lysate; and(e) incubating the reaction mixture in the presence of a cellodextrinand inorganic phosphate to produce ribose.77. A cell-free method for producing ribose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable cellodextrin phosphorylases,thermostable phosphoglucomutases, thermostable glucose 6-phosphatedehydrogenases, thermostable 6-phosphogluconolactonases, thermostable6-phosphogluconate dehydrogenases, thermostable ribose 5-phosphateisomerases, and thermostable ribose 5-phosphate phosphatases to produceat least two cultured populations of cells expressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (a) to produce a heat-inactivated lysate;(e) adding to the heat-inactivated lysate at least one purified enzymeselected from the group consisting of thermostable cellodextrinphosphorylases, thermostable phosphoglucomutases, thermostable glucose6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases,thermostable 6-phosphogluconate dehydrogenases, thermostable ribose5-phosphate isomerases, and thermostable ribose 5-phosphate phosphatasesto produce a reaction mixture comprising a cellodextrin phosphorylase, aphosphoglucomutase, a glucose 6-phosphate dehydrogenase, a6-phosphogluconolactonase, a 6-phosphogluconate dehydrogenase, a ribose5-phosphate isomerase, and a ribose 5-phosphate phosphatase; and(f) incubating the reaction mixture in the presence of a cellodextrinand inorganic phosphate to produce ribose.78. A cell-free method for producing arabinose, the method comprising:(a) culturing cells engineered to express a thermostable cellodextrinphosphorylase, a thermostable phosphoglucomutase, a thermostable glucose6-phosphate dehydrogenase, a thermostable 6-phosphogluconolactonase, athermostable 6-phosphogluconate dehydrogenase, a thermostable arabinose5-phosphate isomerase, and a thermostable arabinose 5-phosphatephosphatase to produce cultured cells that express the thermostableenzymes;(b) lysing the cultured cells to produce a cell lysate;(c) heating the cell lysate to a temperature that inactivates nativeenzymatic activity but does not inactivate the thermostable enzymes ofstep (a) to produce a heat-inactivated lysate; and(d) incubating the heat-inactivated lysate in the presence of acellodextrin and inorganic phosphate to produce arabinose.79. A cell-free method for producing arabinose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable cellodextrin phosphorylases,thermostable phosphoglucomutases, thermostable glucose 6-phosphatedehydrogenases, thermostable 6-phosphogluconolactonases, thermostable6-phosphogluconate dehydrogenases, thermostable arabinose 5-phosphateisomerases, and thermostable arabinose 5-phosphate phosphatases toproduce at least two cultured populations of cells expressing differentenzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture that comprises a thermostable cellodextrin phosphorylase, athermostable phosphoglucomutase, a thermostable glucose 6-phosphatedehydrogenase, a thermostable 6-phosphogluconolactonase, a thermostable6-phosphogluconate dehydrogenase, a thermostable arabinose 5-phosphateisomerase, and a thermostable arabinose 5-phosphate phosphatase;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (c) to produce a heat-inactivated lysate; and(e) incubating the reaction mixture in the presence of a cellodextrinand inorganic phosphate to produce arabinose.80. A cell-free method for producing arabinose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable cellodextrin phosphorylases,thermostable phosphoglucomutases, thermostable glucose 6-phosphatedehydrogenases, thermostable 6-phosphogluconolactonases, thermostable6-phosphogluconate dehydrogenases, thermostable arabinose 5-phosphateisomerases, and thermostable arabinose 5-phosphate phosphatases toproduce at least two cultured populations of cells expressing differentenzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (a) to produce a heat-inactivated lysate;(e) adding to the heat-inactivated lysate at least one purified enzymeselected from the group consisting of thermostable cellodextrinphosphorylases, thermostable phosphoglucomutases, thermostable glucose6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases,thermostable 6-phosphogluconate dehydrogenases, thermostable arabinose5-phosphate isomerases, and thermostable arabinose 5-phosphatephosphatases to produce a reaction mixture comprising a cellodextrinphosphorylase, a phosphoglucomutase, a glucose 6-phosphatedehydrogenase, a 6-phosphogluconolactonase, a 6-phosphogluconatedehydrogenase, an arabinose 5-phosphate isomerase, and an arabinose5-phosphate phosphatase; and(f) incubating the reaction mixture in the presence of a cellodextrinand inorganic phosphate to produce arabinose.81. A cell-free method for producing mannose, the method comprising:(a) culturing cells engineered to express a thermostable cellodextrinphosphorylase, a thermostable phosphoglucomutase, a thermostablephosphoglucoisomerase, a thermostable mannose 6-phosphate epimerase, anda thermostable mannose 6-phosphate phosphatase to produce cultured cellsthat express the thermostable enzymes;(b) lysing the cultured cells to produce a cell lysate;(c) heating the cell lysate to a temperature that inactivates nativeenzymatic activity but does not inactivate the thermostable enzymes ofstep (a) to produce a heat-inactivated lysate; and(d) incubating the heat-inactivated lysate in the presence of acellodextrin and inorganic phosphate to produce mannose.82. A cell-free method for producing mannose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable cellodextrin phosphorylases,thermostable phosphoglucomutases, thermostable phosphoglucoisomerases,thermostable mannose 6-phosphate epimerases, and thermostable mannose6-phosphate phosphatases to produce at least two cultured populations ofcells expressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture that comprises a thermostable cellodextrin phosphorylase, athermostable phosphoglucomutase, a thermostable phosphoglucoisomerase, athermostable mannose 6-phosphate epimerase, and a thermostable mannose6-phosphate phosphatase;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (c) to produce a heat-inactivated lysate; and(e) incubating the reaction mixture in the presence of a cellodextrinand inorganic phosphate to produce mannose.83. A cell-free method for producing mannose, the method comprising:(a) culturing at least two cell populations, wherein cells of eachpopulation are engineered to express at least one enzyme selected fromthe group consisting of thermostable cellodextrin phosphorylases,thermostable phosphoglucomutases, thermostable phosphoglucoisomerases,thermostable mannose 6-phosphate epimerases, and thermostable mannose6-phosphate phosphatases to produce at least two cultured populations ofcells expressing different enzymes;(b) lysing cells of the at least two cultured populations to produce atleast two cell lysates;(c) combining the at least two cell lysates to produce a cell lysatemixture;(d) heating the cell lysate mixture to a temperature that inactivatesnative enzymatic activity but does not inactivate the thermostableenzymes of step (a) to produce a heat-inactivated lysate;(e) adding to the heat-inactivated lysate at least one purified enzymeselected from the group consisting of thermostable cellodextrinphosphorylases, thermostable phosphoglucomutases, thermostablephosphoglucoisomerases, thermostable mannose 6-phosphate epimerases, andthermostable mannose 6-phosphate phosphatases to produce a reactionmixture comprising a cellodextrin phosphorylase, a phosphoglucomutase, aphosphoglucoisomerase, an mannose 6-phosphate epimerase, and an mannose6-phosphate phosphatase; and(f) incubating the reaction mixture in the presence of a cellodextrinand inorganic phosphate to produce mannose.84. The method of any one of embodiments 1-80, wherein the cellscomprise bacterial cells.85. The method of any one of embodiments 1-80, wherein the cellscomprise yeast cells.86. The method of any one of embodiments 1-85, wherein at least one ofthe enzymes is heterologous to the cells.87. The method of any one of embodiments 1-86, wherein lysing step (b)comprises mechanically, chemically, or enzymatically lysing the culturedcells.88. The method for any one of embodiments 1-87, wherein heating step (c)comprises heating the cell lysate to a temperature of at least 50° C.89. The method for any one of embodiments 1-88, wherein the cellodextrincomprises amylose, amylopectin, or both amylose and amylopectin.90. The method for any one of embodiments 1-89, wherein the thermostablecellodextrin phosphorylase and the thermostable phosphoglucomutase areexpressed as a single fusion protein or a bifunctional protein.91. A cell lysate produced by the method for any one of embodiments1-90.92. An engineered cell comprising a cellodextrin phosphorylase, aphosphoglucomutase, and a glucose 6-phosphate phosphatase, optionallywherein at least one of the foregoing enzymes is a thermostable enzyme.93. An engineered cell comprising a cellodextrin phosphorylase, aphosphoglucomutase, a phosphoglucoisomerase, and a fructose 6-phosphatephosphatase, optionally wherein at least one of the foregoing enzymes isa thermostable enzyme.94. An engineered cell comprising a cellodextrin phosphorylase, aphosphoglucomutase, a phosphoglucoisomerase, an allulose 6-phosphateepimerase, and an allulose 6-phosphate phosphatase, optionally whereinat least one of the foregoing enzymes is a thermostable enzyme.95. An engineered cell comprising a cellodextrin phosphorylase, aphosphoglucomutase, a aldose dehydrogenase, and a sorbitol-6-phosphatephosphatase, optionally wherein at least one of the foregoing enzymes isa thermostable enzyme.96. An engineered cell comprising a cellodextrin phosphorylase, aphosphoglucomutase, a glucose 6-phosphate dehydrogenase, a6-phosphogluconolactonase, a 6-phosphogluconate dehydrogenase, and aribulose 5-phosphate phosphatase, optionally wherein at least one of theforegoing enzymes is a thermostable enzyme.97. An engineered cell comprising a cellodextrin phosphorylase, aphosphoglucomutase, a glucose 6-phosphate dehydrogenase, a6-phosphogluconolactonase, a 6-phosphogluconate dehydrogenase, a ribose5-phosphate isomerase, and a ribose 5-phosphate phosphatase, optionallywherein at least one of the foregoing enzymes is a thermostable enzyme.98. An engineered cell comprising a cellodextrin phosphorylase, aphosphoglucomutase, a glucose 6-phosphate dehydrogenase, a6-phosphogluconolactonase, a 6-phosphogluconate dehydrogenase, anarabinose 5-phosphate isomerase, and an arabinose 5-phosphatephosphatase, optionally wherein at least one of the foregoing enzymes isa thermostable enzyme.99. An engineered cell comprising a cellodextrin phosphorylase, aphosphoglucomutase, a phosphoglucoisomerase, an mannose 6-phosphateepimerase, and an mannose 6-phosphate phosphatase, optionally wherein atleast one of the foregoing enzymes is a thermostable enzyme.100. The engineered cell of any one of embodiments embodiment 35-41,wherein the cell is a bacterial cell or a yeast cell.101. A single cell lysate, a mixture of cell lysates obtained from atleast two cell populations, or a reaction mixture comprising acellodextrin phosphorylase, a phosphoglucomutase, and a glucose6-phosphate phosphatase, optionally wherein at least one of theforegoing enzymes is a thermostable enzyme.102. A single cell lysate, a mixture of cell lysates obtained from atleast two cell populations, or a reaction mixture comprising acellodextrin phosphorylase, a phosphoglucomutase, aphosphoglucoisomerase, and a fructose 6-phosphate phosphatase,optionally wherein at least one of the foregoing enzymes is athermostable enzyme.103. A single cell lysate, a mixture of cell lysates obtained from atleast two cell populations, or a reaction mixture comprising acellodextrin phosphorylase, a phosphoglucomutase, aphosphoglucoisomerase, an allulose 6-phosphate epimerase, and anallulose 6-phosphate phosphatase, optionally wherein at least one of theforegoing enzymes is a thermostable enzyme.104. A single cell lysate, a mixture of cell lysates obtained from atleast two cell populations, or a reaction mixture comprising acellodextrin phosphorylase, a phosphoglucomutase, a aldosedehydrogenase, and a sorbitol-6-phosphate phosphatase, optionallywherein at least one of the foregoing enzymes is a thermostable enzyme.105. A single cell lysate, a mixture of cell lysates obtained from atleast two cell populations, or a reaction mixture comprising acellodextrin phosphorylase, a phosphoglucomutase, a glucose 6-phosphatedehydrogenase, a 6-phosphogluconolactonase, a 6-phosphogluconatedehydrogenase, and a ribulose 5-phosphate phosphatase, optionallywherein at least one of the foregoing enzymes is a thermostable enzyme.106. A single cell lysate, a mixture of cell lysates obtained from atleast two cell populations, or a reaction mixture comprising acellodextrin phosphorylase, a phosphoglucomutase, a glucose 6-phosphatedehydrogenase, a 6-phosphogluconolactonase, a 6-phosphogluconatedehydrogenase, a ribose 5-phosphate isomerase, and a ribose 5-phosphatephosphatase, optionally wherein at least one of the foregoing enzymes isa thermostable enzyme.107. A single cell lysate, a mixture of cell lysates obtained from atleast two cell populations, or a reaction mixture comprising acellodextrin phosphorylase, a phosphoglucomutase, a glucose 6-phosphatedehydrogenase, a 6-phosphogluconolactonase, a 6-phosphogluconatedehydrogenase, an arabinose 5-phosphate isomerase, and an arabinose5-phosphate phosphatase, optionally wherein at least one of theforegoing enzymes is a thermostable enzyme.108. A single cell lysate, a mixture of cell lysates obtained from atleast two cell populations, or a reaction mixture comprising acellodextrin phosphorylase, a phosphoglucomutase, aphosphoglucoisomerase, an mannose 6-phosphate epimerase, and an mannose6-phosphate phosphatase, optionally wherein at least one of theforegoing enzymes is a thermostable enzyme.109. A cell-free method for producing a sugar, the method comprising:

(a) culturing cells engineered to express a thermostable α-glucanphosphorylase, a thermostable phosphoglucomutase, and at least onethermostable enzyme selected from the group consisting of isomerases,epimerases, dehydrogenases, and sugar phosphatases to produce culturedcells that express the enzymes;

(b) lysing cultured cells of step (a) to produce a cell lysate;

(c) heating the cell lysate to a temperature that inactivates nativeenzymatic activity but does not inactivate the thermostable enzymes ofstep (a) to produce a heat-inactivated lysate; and

(d) incubating the heat-inactivated lysate in the presence of a starchand inorganic phosphate to produce the sugar.

110. The method for embodiment 109, wherein the at least onethermostable enzyme is an isomerase selected from the group consistingof: phosphoglucoisomerase, ribose 5-phosphate isomerase, and arabinose5-phosphate isomerase.

111. The method for embodiment 109 or 110, wherein the at least onethermostable enzyme is allulose 6-phosphate epimerase.

112. The method for any one of embodiments 109-111, wherein the at leastone thermostable enzyme is a dehydrogenase selected from the groupconsisting of aldose dehydrogenase, glucose 6-phosphate dehydrogenase,and 6-phosphogluconate dehydrogenase.

113. The method for any one of embodiments 109-112, wherein the at leastone thermostable enzyme is a sugar phosphatase selected from the groupconsisting of glucose 6-phosphate phosphatase, fructose 6-phosphatephosphatase, allulose 6-phosphate phosphatase, sorbitol-6-phosphatephosphatase, ribulose 5-phosphate phosphatase, ribose 5-phosphatephosphatase, and arabinose 5-phosphate phosphatase.114. The method for embodiment 109, wherein the sugar is selected fromthe group consisting of glucose, fructose, allulose, sorbitol, ribulose,ribose, and arabinose.115. The method for embodiment 114, wherein the sugar is glucose, andthe at least one thermostable enzyme comprises glucose 6-phosphatephosphatase.116. The method of embodiment 115, wherein the cells are engineered toexpress a thermostable α-glucan phosphorylase, a thermostablephosphoglucomutase, and a thermostable glucose 6-phosphate phosphatase.117. The method for embodiment 114, wherein the sugar is fructose, andthe at least one thermostable enzyme is selected fromphosphoglucoisomerases and fructose 6-phosphate phosphatases.118. The method for embodiment 117, wherein the cells are engineered toexpress a thermostable α-glucan phosphorylase, a thermostablephosphoglucomutase, a thermostable phosphoglucoisomerase, and athermostable fructose 6-phosphate phosphatase.119. The method for embodiment 114, wherein the sugar is allulose, andthe at least one thermostable enzyme is selected fromphosphoglucoisomerases, allulose 6-phosphate epimerases, and allulose6-phosphate phosphatases.120. The method for embodiment 119, wherein the cells are engineered toexpress a thermostable α-glucan phosphorylase, a thermostablephosphoglucomutase, a thermostable phosphoglucoisomerase, a thermostableallulose 6-phosphate epimerase, and a thermostable allulose 6-phosphatephosphatase.121. The method for embodiment 114, wherein the sugar is sorbitol, andthe at least one thermostable enzyme is selected from aldosedehydrogenases and sorbitol-6-phosphate phosphatases.122. The method for embodiment 121, wherein the cells are engineered toexpress a thermostable α-glucan phosphorylase, a thermostablephosphoglucomutase, a thermostable aldose dehydrogenase, and athermostable sorbitol-6-phosphate phosphatase.123. The method for embodiment 114, wherein the sugar is ribulose, andthe at least one thermostable enzyme is selected from glucose6-phosphate dehydrogenases, 6-phosphogluconolactonases,6-phosphogluconate dehydrogenases, and ribulose 5-phosphatephosphatases.124. The method for embodiment 123, wherein the cells are engineered toexpress a thermostable α-glucan phosphorylase, a thermostablephosphoglucomutase, a thermostable glucose 6-phosphate dehydrogenase, athermostable 6-phosphogluconolactonase, a thermostable6-phosphogluconate dehydrogenase, and a thermostable ribulose5-phosphate phosphatase.125. The method for embodiment 114, wherein the sugar is ribose, and theat least one thermostable enzyme is selected from glucose 6-phosphatedehydrogenases, 6-phosphogluconolactonases, 6-phosphogluconatedehydrogenases, ribose 5-phosphate isomerases, and ribose 5-phosphatephosphatases.126. The method for embodiment 125, wherein the cells are engineered toexpress a thermostable α-glucan phosphorylase, a thermostablephosphoglucomutase, a thermostable glucose 6-phosphate dehydrogenase, athermostable 6-phosphogluconolactonase, a thermostable6-phosphogluconate dehydrogenase, a thermostable ribose 5-phosphateisomerase, and a thermostable ribose 5-phosphate phosphatase.127. The method for embodiment 114, wherein the sugar is arabinose, andthe at least one thermostable enzyme is selected from glucose6-phosphate dehydrogenases, 6-phosphogluconolactonases,6-phosphogluconate dehydrogenases, arabinose 5-phosphate isomerases, andarabinose 5-phosphate phosphatases.128. The method for embodiment 127, wherein the cells are engineered toexpress a thermostable α-glucan phosphorylase, a thermostablephosphoglucomutase, a thermostable glucose 6-phosphate dehydrogenase, athermostable 6-phosphogluconolactonase, a thermostable6-phosphogluconate dehydrogenase, a thermostable arabinose 5-phosphateisomerase, and a thermostable arabinose 5-phosphate phosphatase.129. The method for any one of embodiments 109-128, wherein the cellsare bacterial cells.130. The method for any one of embodiments 109-128, wherein the cellsare yeast cells.131. The method for any one of embodiments 109-130, wherein thethermostable α-glucan phosphorylase, the thermostablephosphoglucomutase, and/or the at least one thermostable enzyme is/areheterologous to the cells.132. The method for any one of embodiments 109-131, wherein lysing step(b) comprises mechanically, chemically, or enzymatically lysing thecultured cells.133. The method for any one of embodiments 109-132, wherein heating step(c) comprises heating the cell lysate to a temperature of at least 50°C.134. The method for any one of embodiments 109-133, wherein the starchcomprises amylose, amylopectin, or both amylose and amylopectin.135. The method for any one of embodiments 109-134, wherein thethermostable α-glucan phosphorylase and the thermostablephosphoglucomutase are expressed as a single fusion protein.136. A cell-free method for producing a sugar, the method comprising:(a) culturing cells engineered to express a α-glucan phosphorylase, aphosphoglucomutase, and at least one enzyme selected from the groupconsisting of isomerases, epimerases, dehydrogenases, and sugarphosphatases to produce cultured cells that express the enzymes;(b) lysing cultured cells of step (a) to produce a cell lysate; and(c) incubating the lysate in the presence of a starch and inorganicphosphate to produce the sugar.137. A cell-free method for producing a sugar, the method comprising:(a) culturing cells engineered to express (ii) a fusion protein thatcomprises a α-glucan phosphorylase fused to a phosphoglucomutase, and(ii) at least one enzyme selected from the group consisting ofisomerases, epimerases, dehydrogenases, and sugar phosphatases toproduce cultured cells that express the enzymes;(b) lysing cultured cells of step (a) to produce a cell lysate; and(c) incubating the lysate in the presence of a starch and inorganicphosphate to produce the sugar.138. A cell-free method for producing a sugar, the method comprising:(a) culturing cells engineered to express a fusion protein thatcomprises a α-glucan phosphorylase fused to a phosphoglucomutase;(b) culturing cells engineered to express at least one enzyme selectedfrom the group consisting of isomerases, epimerases, dehydrogenases, andsugar phosphatases to produce cultured cells that express the enzymes;(c) lysing cultured cells of step (a) and step (b) to produce celllysates; and(d) incubating the lysates in the presence of a starch and inorganicphosphate to produce the sugar.139. The method for embodiment 137 or 138, wherein the enzymes of steps(a) and/or (b) are thermostable enzymes.140. The method for embodiment 139, wherein the method further comprisesheating the cell lysate(s) to a temperature that inactivates nativeenzymatic activity but does not inactivate the thermostable enzymes toproduce heat-inactivated lysate(s).141. A cell lysate produced by the method for any one of embodiments109-138.142. An engineered cell comprising a thermostable α-glucanphosphorylase, a thermostable phosphoglucomutase, and at least onethermostable enzyme selected from the group consisting of isomerases,epimerases, dehydrogenases, and sugar phosphatases.143. The engineered cell of embodiment 142 comprising:(a) a thermostable α-glucan phosphorylase, a thermostablephosphoglucomutase, and a thermostable glucose 6-phosphate phosphatase;(b) a thermostable α-glucan phosphorylase, a thermostablephosphoglucomutase, a phosphoglucoisomerase, and a fructose 6-phosphatephosphatase;(c) a thermostable α-glucan phosphorylase, a thermostablephosphoglucomutase, a phosphoglucoisomerase, an allulose 6-phosphateepimerase, and an allulose 6-phosphate phosphatase;(d) a thermostable α-glucan phosphorylase, a thermostablephosphoglucomutase, an aldose dehydrogenase, and a sorbitol-6-phosphatephosphatase;(e) a thermostable α-glucan phosphorylase, a thermostablephosphoglucomutase, a glucose 6-phosphate dehydrogenase, a6-phosphogluconolactonase, a 6-phosphogluconate dehydrogenase, and aribulose 5-phosphate phosphatase;(f) a thermostable α-glucan phosphorylase, a thermostablephosphoglucomutase, a glucose 6-phosphate dehydrogenase, a6-phosphogluconolactonase, a 6-phosphogluconate dehydrogenase, a ribose5-phosphate isomerase, and a ribose 5-phosphate phosphatase; or(g) a thermostable α-glucan phosphorylase, a thermostablephosphoglucomutase, a glucose 6-phosphate dehydrogenase, a6-phosphogluconolactonase, a 6-phosphogluconate dehydrogenase, anarabinose 5-phosphate isomerase, and an arabinose 5-phosphatephosphatase.144. The engineered cell of embodiment 142 or 143, wherein the cell is abacterial cell or a yeast cell.

EXAMPLES Example 1. Cell Free Conversion of Starch to Allulose

This example describes the conversion of starch to allulose. Cells(e.g., bacterial or yeast cells) engineered to express at least oneheterologous genes encoding at least one enzyme for the conversion ofstarch to allulose are grown in liquid cultures to high cell density.Examples of heterologous enzymes that may be used in this exampleinclude thermostable variants of a α-glucan phosphorylase (EC 2.4.1.1),a phosphoglucomutase (EC 5.4.2.2, 5.4.2.5, or 5.4.2.6), aphosphoglucoisomerase (EC 5.3.1.9), an allulose 6-phosphate epimerase(EC 5.3.1.-), and an allulose 6-phosphate phosphatase (EC 5.3.1.-). Atthe end of the growth stage, expression of the heterologous enzyme(s) isinduced, and the cell biomass is subsequently harvested. The harvestedbiomass is then lysed via mechanical, chemical or enzymatic means. Thecell lysate is then heated to a temperature that inactivates nativeenzymatic activities but does not inactivate the heterologous enzyme(s).A starch feedstock, inorganic phosphate and optionally other additionalnutrients are added to the heat inactivated lysate to enable conversionof starch to allulose (FIG. 1).

Example 2. Cell Free Conversion of Starch to Glucose

This example describes the conversion of starch to glucose. Cells (e.g.,bacterial or yeast cells) engineered to express at least oneheterologous genes encoding at least one enzyme for the conversion ofstarch to glucose are grown in liquid cultures to high cell density.Examples of heterologous enzymes that may be used in this exampleinclude thermostable variants of a α-glucan phosphorylase (EC 2.4.1.1),a phosphoglucomutase (EC 5.4.2.2, 5.4.2.5, or 5.4.2.6) and a glucose6-phosphate phosphatase (EC 5.3.1.-). At the end of the growth stage,expression of the heterologous enzyme(s) is induced, and the cellbiomass is subsequently harvested. The harvested biomass is then lysedvia mechanical, chemical or enzymatic means. The cell lysate is thenheated to a temperature that inactivates native enzymatic activities butdoes not inactivate the heterologous enzyme(s). A starch feedstock,inorganic phosphate and optionally other additional nutrients are addedto the heat inactivated lysate to enable conversion of starch to glucose(FIG. 2A).

This example also describes another pathway for the conversion of starchto glucose. Cells (e.g., bacterial or yeast cells) engineered to expressat least one heterologous genes encoding at least one enzyme for theconversion of starch to glucose are grown in liquid cultures to highcell density. Examples of heterologous enzymes that may be used in thisexample include thermostable variants of a α-glucan phosphorylase (EC2.4.1.1), and a glucose 1-phosphate phosphatase (EC 3.1.3.10). At theend of the growth stage, expression of the heterologous enzyme(s) isinduced, and the cell biomass is subsequently harvested. The harvestedbiomass is then lysed via mechanical, chemical or enzymatic means. Thecell lysate is then heated to a temperature that inactivates nativeenzymatic activities but does not inactivate the heterologous enzyme(s).A starch feedstock, inorganic phosphate and optionally other additionalnutrients are added to the heat inactivated lysate to enable conversionof starch to glucose (FIG. 2B).

Example 3. Cell Free Conversion of Starch to Fructose

This example describes the conversion of starch to fructose. Cells(e.g., bacterial or yeast cells) engineered to express at least oneheterologous genes encoding at least one enzyme for the conversion ofstarch to fructose are grown in liquid cultures to high cell density.Examples of heterologous enzymes that may be used in this exampleinclude thermostable variants of a α-glucan phosphorylase (EC 2.4.1.1),a phosphoglucomutase (EC 5.4.2.2, 5.4.2.5, or 5.4.2.6), aphosphoglucoisomerase (EC 5.3.1.9), and a fructose 6-phosphatephosphatase (EC 5.3.1.-). At the end of the growth stage, expression ofthe heterologous enzyme(s) is induced, and the cell biomass issubsequently harvested. The harvested biomass is then lysed viamechanical, chemical or enzymatic means. The cell lysate is then heatedto a temperature that inactivates native enzymatic activities but doesnot inactivate the heterologous enzyme(s). A starch feedstock, inorganicphosphate and optionally other additional nutrients are added to theheat inactivated lysate to enable conversion of starch to fructose (FIG.3).

Example 4. Cell Free Conversion of Starch to Sorbitol

This example describes the conversion of starch to sorbitol. Cells(e.g., bacterial or yeast cells) engineered to express at least oneheterologous genes encoding at least one enzyme for the conversion ofstarch to sorbitol are grown in liquid cultures to high cell density.Examples of heterologous enzymes that may be used in this exampleinclude thermostable variants of a α-glucan phosphorylase (EC 2.4.1.1),a phosphoglucomutase (EC 5.4.2.2, 5.4.2.5, or 5.4.2.6), an aldosedehydrogenase (EC 1.1.1.200), and a sorbitol-6-phosphate phosphatase (EC5.3.1.-). At the end of the growth stage, expression of the heterologousenzyme(s) is induced, and the cell biomass is subsequently harvested.The harvested biomass is then lysed via mechanical, chemical orenzymatic means. The cell lysate is then heated to a temperature thatinactivates native enzymatic activities but does not inactivate theheterologous enzyme(s). A starch feedstock, inorganic phosphate andoptionally other additional nutrients are added to the heat inactivatedlysate to enable conversion of starch to sorbitol (FIG. 4).

Example 5. Cell Free Conversion of Starch to Ribulose

This example describes the conversion of starch to ribulose. Cells(e.g., bacterial or yeast cells) engineered to express at least oneheterologous genes encoding at least one enzyme for the conversion ofstarch to ribulose are grown in liquid cultures to high cell density.Examples of heterologous enzymes that may be used in this exampleinclude thermostable variants of a α-glucan phosphorylase (EC 2.4.1.1),a phosphoglucomutase (EC 5.4.2.2, 5.4.2.5, or 5.4.2.6), a glucose6-phosphate dehydrogenase (EC 1.1.1.49), a 6-phosphogluconolactonase (EC3.1.1.31), a 6-phosphogluconate dehydrogenase (EC 1.1.1.44), and aribulose 5-phosphate phosphatase (EC 5.3.1.-), and an ribulose6-phosphate phosphatase (EC 5.3.1.-). At the end of the growth stage,expression of the heterologous enzyme(s) is induced, and the cellbiomass is subsequently harvested. The harvested biomass is then lysedvia mechanical, chemical or enzymatic means. The cell lysate is thenheated to a temperature that inactivates native enzymatic activities butdoes not inactivate the heterologous enzyme(s). A starch feedstock,inorganic phosphate and optionally other additional nutrients are addedto the heat inactivated lysate to enable conversion of starch toribulose (FIG. 5).

Example 6. Cell Free Conversion of Starch to Ribose

This example describes the conversion of starch to ribose. Cells (e.g.,bacterial or yeast cells) engineered to express at least oneheterologous genes encoding at least one enzyme for the conversion ofstarch to ribose are grown in liquid cultures to high cell density.Examples of heterologous enzymes that may be used in this exampleinclude thermostable variants of a α-glucan phosphorylase (EC 2.4.1.1),a phosphoglucomutase (EC 5.4.2.2, 5.4.2.5, or 5.4.2.6), a glucose6-phosphate dehydrogenase (EC 1.1.1.49), a 6-phosphogluconolactonase (EC3.1.1.31), a 6-phosphogluconate dehydrogenase (EC 1.1.1.44), a ribose5-phosphate isomerase (EC 5.3.1.6) and a ribose 5-phosphate phosphatase(EC 5.3.1.-). At the end of the growth stage, expression of theheterologous enzyme(s) is induced, and the cell biomass is subsequentlyharvested. The harvested biomass is then lysed via mechanical, chemicalor enzymatic means. The cell lysate is then heated to a temperature thatinactivates native enzymatic activities but does not inactivate theheterologous enzyme(s). A starch feedstock, inorganic phosphate andoptionally other additional nutrients are added to the heat inactivatedlysate to enable conversion of starch to ribose (FIG. 6).

Example 7. Cell Free Conversion of Starch to Arabinose

This example describes the conversion of starch to arabinose. Cells(e.g., bacterial or yeast cells) engineered to express at least oneheterologous genes encoding at least one enzyme for the conversion ofstarch to arabinose are grown in liquid cultures to high cell density.Examples of heterologous enzymes that may be used in this exampleinclude thermostable variants of a α-glucan phosphorylase (EC 2.4.1.1),a phosphoglucomutase (EC 5.4.2.2, 5.4.2.5, or 5.4.2.6), a glucose6-phosphate dehydrogenase (EC 1.1.1.49), a 6-phosphogluconolactonase (EC3.1.1.31), a 6-phosphogluconate dehydrogenase (EC 1.1.1.44), anarabinose 5-phosphate isomerase (EC 5.3.1.6) and an arabinose5-phosphate phosphatase (EC 5.3.1.-). At the end of the growth stage,expression of the heterologous enzyme(s) is induced, and the cellbiomass is subsequently harvested. The harvested biomass is then lysedvia mechanical, chemical or enzymatic means. The cell lysate is thenheated to a temperature that inactivates native enzymatic activities butdoes not inactivate the heterologous enzyme(s). A starch feedstock,inorganic phosphate and optionally other additional nutrients are addedto the heat inactivated lysate to enable conversion of starch toarabinose (FIG. 7).

Example 8. Cell Free Conversion of Cellulose/Cellodextrin to Allulose

This example describes the conversion of cellulose/cellodextrin toallulose. Cells (e.g., bacterial or yeast cells) engineered to expressat least one heterologous genes encoding at least one enzyme for theconversion of cellulose/cellodextrin to allulose are grown in liquidcultures to high cell density. Examples of heterologous enzymes that maybe used in this example include thermostable variants of a cellodextrinphosphorylase (EC 2.4.1.49), a phosphoglucomutase (EC 5.4.2.2, 5.4.2.5,or 5.4.2.6), a phosphoglucoisomerase (EC 5.3.1.9), an allulose6-phosphate epimerase (EC 5.3.1.-), and an allulose 6-phosphatephosphatase (EC 5.3.1.-). At the end of the growth stage, expression ofthe heterologous enzyme(s) is induced, and the cell biomass issubsequently harvested. The harvested biomass is then lysed viamechanical, chemical or enzymatic means. The cell lysate is then heatedto a temperature that inactivates native enzymatic activities but doesnot inactivate the heterologous enzyme(s). A cellulose/cellodextrinfeedstock, inorganic phosphate and optionally other additional nutrientsare added to the heat inactivated lysate to enable conversion ofcellulose/cellodextrin to allulose.

Example 9. Cell Free Conversion of Cellulose/Cellodextrin to Glucose

This example describes the conversion of cellulose/cellodextrin toglucose. Cells (e.g., bacterial or yeast cells) engineered to express atleast one heterologous genes encoding at least one enzyme for theconversion of cellulose/cellodextrin to glucose are grown in liquidcultures to high cell density. Examples of heterologous enzymes that maybe used in this example include thermostable variants of a cellodextrinphosphorylase (EC 2.4.1.49), a phosphoglucomutase (EC 5.4.2.2, 5.4.2.5,or 5.4.2.6) and a glucose 6-phosphate phosphatase (EC 5.3.1.-). At theend of the growth stage, expression of the heterologous enzyme(s) isinduced, and the cell biomass is subsequently harvested. The harvestedbiomass is then lysed via mechanical, chemical or enzymatic means. Thecell lysate is then heated to a temperature that inactivates nativeenzymatic activities but does not inactivate the heterologous enzyme(s).A cellulose/cellodextrin feedstock, inorganic phosphate and optionallyother additional nutrients are added to the heat inactivated lysate toenable conversion of cellulose/cellodextrin to glucose.

This example also describes another pathway for the conversion ofcellulose/cellodextrin to glucose. Cells (e.g., bacterial or yeastcells) engineered to express at least one heterologous genes encoding atleast one enzyme for the conversion of cellulose/cellodextrin to glucoseare grown in liquid cultures to high cell density. Examples ofheterologous enzymes that may be used in this example includethermostable variants of a cellodextrin phosphorylase (EC 2.4.1.49), anda glucose 1-phosphate phosphatase (EC 3.1.3.10). At the end of thegrowth stage, expression of the heterologous enzyme(s) is induced, andthe cell biomass is subsequently harvested. The harvested biomass isthen lysed via mechanical, chemical or enzymatic means. The cell lysateis then heated to a temperature that inactivates native enzymaticactivities but does not inactivate the heterologous enzyme(s). Acellulose/cellodextrin feedstock, inorganic phosphate and optionallyother additional nutrients are added to the heat inactivated lysate toenable conversion of cellulose/cellodextrin to glucose.

Example 10. Cell Free Conversion of Cellulose/Cellodextrin to Fructose

This example describes the conversion of cellulose/cellodextrin tofructose. Cells (e.g., bacterial or yeast cells) engineered to expressat least one heterologous genes encoding at least one enzyme for theconversion of cellulose/cellodextrin to fructose are grown in liquidcultures to high cell density. Examples of heterologous enzymes that maybe used in this example include thermostable variants of a cellodextrinphosphorylase (EC 2.4.1.49), a phosphoglucomutase (EC 5.4.2.2, 5.4.2.5,or 5.4.2.6), a phosphoglucoisomerase (EC 5.3.1.9), and a fructose6-phosphate phosphatase (EC 5.3.1.-). At the end of the growth stage,expression of the heterologous enzyme(s) is induced, and the cellbiomass is subsequently harvested. The harvested biomass is then lysedvia mechanical, chemical or enzymatic means. The cell lysate is thenheated to a temperature that inactivates native enzymatic activities butdoes not inactivate the heterologous enzyme(s). A cellulose/cellodextrinfeedstock, inorganic phosphate and optionally other additional nutrientsare added to the heat inactivated lysate to enable conversion ofcellulose/cellodextrin to fructose.

Example 11. Cell Free Conversion of Cellulose/Cellodextrin to Sorbitol

This example describes the conversion of cellulose/cellodextrin tosorbitol. Cells (e.g., bacterial or yeast cells) engineered to expressat least one heterologous genes encoding at least one enzyme for theconversion of cellulose/cellodextrin to sorbitol are grown in liquidcultures to high cell density. Examples of heterologous enzymes that maybe used in this example include thermostable variants of a cellodextrinphosphorylase (EC 2.4.1.49), a phosphoglucomutase (EC 5.4.2.2, 5.4.2.5,or 5.4.2.6), an aldose dehydrogenase (EC 1.1.1.200), and asorbitol-6-phosphate phosphatase (EC 5.3.1.-). At the end of the growthstage, expression of the heterologous enzyme(s) is induced, and the cellbiomass is subsequently harvested. The harvested biomass is then lysedvia mechanical, chemical or enzymatic means. The cell lysate is thenheated to a temperature that inactivates native enzymatic activities butdoes not inactivate the heterologous enzyme(s). A cellulose/cellodextrinfeedstock, inorganic phosphate and optionally other additional nutrientsare added to the heat inactivated lysate to enable conversion ofcellulose/cellodextrin to sorbitol.

Example 12. Cell Free Conversion of Cellulose/Cellodextrin to Ribulose

This example describes the conversion of cellulose/cellodextrin toribulose. Cells (e.g., bacterial or yeast cells) engineered to expressat least one heterologous genes encoding at least one enzyme for theconversion of cellulose/cellodextrin to ribulose are grown in liquidcultures to high cell density. Examples of heterologous enzymes that maybe used in this example include thermostable variants of a cellodextrinphosphorylase (EC 2.4.1.49), a phosphoglucomutase (EC 5.4.2.2, 5.4.2.5,or 5.4.2.6), a glucose 6-phosphate dehydrogenase (EC 1.1.1.49), a6-phosphogluconolactonase (EC 3.1.1.31), a 6-phosphogluconatedehydrogenase (EC 1.1.1.44), and a ribulose 5-phosphate phosphatase (EC5.3.1.-), and an ribulose 6-phosphate phosphatase (EC 5.3.1.-). At theend of the growth stage, expression of the heterologous enzyme(s) isinduced, and the cell biomass is subsequently harvested. The harvestedbiomass is then lysed via mechanical, chemical or enzymatic means. Thecell lysate is then heated to a temperature that inactivates nativeenzymatic activities but does not inactivate the heterologous enzyme(s).A cellulose/cellodextrin feedstock, inorganic phosphate and optionallyother additional nutrients are added to the heat inactivated lysate toenable conversion of cellulose/cellodextrin to ribulose.

Example 13. Cell Free Conversion of Cellulose/Cellodextrin to Ribose

This example describes the conversion of cellulose/cellodextrin toribose. Cells (e.g., bacterial or yeast cells) engineered to express atleast one heterologous genes encoding at least one enzyme for theconversion of cellulose/cellodextrin to ribose are grown in liquidcultures to high cell density. Examples of heterologous enzymes that maybe used in this example include thermostable variants of a cellodextrinphosphorylase (EC 2.4.1.49), a phosphoglucomutase (EC 5.4.2.2, 5.4.2.5,or 5.4.2.6), a glucose 6-phosphate dehydrogenase (EC 1.1.1.49), a6-phosphogluconolactonase (EC 3.1.1.31), a 6-phosphogluconatedehydrogenase (EC 1.1.1.44), a ribose 5-phosphate isomerase (EC 5.3.1.6)and a ribose 5-phosphate phosphatase (EC 5.3.1.-). At the end of thegrowth stage, expression of the heterologous enzyme(s) is induced, andthe cell biomass is subsequently harvested. The harvested biomass isthen lysed via mechanical, chemical or enzymatic means. The cell lysateis then heated to a temperature that inactivates native enzymaticactivities but does not inactivate the heterologous enzyme(s). Acellulose/cellodextrin feedstock, inorganic phosphate and optionallyother additional nutrients are added to the heat inactivated lysate toenable conversion of cellulose/cellodextrin to ribose.

Example 14. Cell Free Conversion of Cellulose/Cellodextrin to Arabinose

This example describes the conversion of cellulose/cellodextrin toarabinose. Cells (e.g., bacterial or yeast cells) engineered to expressat least one heterologous genes encoding at least one enzyme for theconversion of cellulose/cellodextrin to arabinose are grown in liquidcultures to high cell density. Examples of heterologous enzymes that maybe used in this example include thermostable variants of a cellodextrinphosphorylase (EC 2.4.1.49), a phosphoglucomutase (EC 5.4.2.2, 5.4.2.5,or 5.4.2.6), a glucose 6-phosphate dehydrogenase (EC 1.1.1.49), a6-phosphogluconolactonase (EC 3.1.1.31), a 6-phosphogluconatedehydrogenase (EC 1.1.1.44), an arabinose 5-phosphate isomerase (EC5.3.1.6) and an arabinose 5-phosphate phosphatase (EC 5.3.1.-). At theend of the growth stage, expression of the heterologous enzyme(s) isinduced, and the cell biomass is subsequently harvested. The harvestedbiomass is then lysed via mechanical, chemical or enzymatic means. Thecell lysate is then heated to a temperature that inactivates nativeenzymatic activities but does not inactivate the heterologous enzyme(s).A cellulose/cellodextrin feedstock, inorganic phosphate and optionallyother additional nutrients are added to the heat inactivated lysate toenable conversion of cellulose/cellodextrin to arabinose.

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited. In the claims, as well as in the specification above, alltransitional phrases such as “comprising,” “including,” “carrying,”“having,” “containing,” “involving,” “holding,” “composed of,” and thelike are to be understood to be open-ended, i.e., to mean including butnot limited to. Only the transitional phrases “consisting of” and“consisting essentially of” shall be closed or semi-closed transitionalphrases, respectively, as set forth in the United States Patent OfficeManual of Patent Examining Procedures, Section 2111.03.

What is claimed is:
 1. A method for producing allulose, the methodcomprising: converting fructose 6-phosphate (F6P) to allulose6-phosphate (A6P), catalyzed by an allulose 6-phosphate epimerase (A6PE)selected from the group consisting of Thermoanaerobacteriumthermosaccharolyticum A6PE, Hydrogenivirga sp. 128-5-R1-1 A6PE, andBrevibacillus thermoruber A6PE; and converting A6P to allulose,catalyzed by Ruminiclostridium thermocellum allulose 6-phosphatephosphatase (A6PP).
 2. The method of claim 1, wherein the A6PE is aThermoanaerobacterium thermosaccharolyticum A6PE.
 3. The method of claim1, wherein the A6PE is a Hydrogenivirga sp. 128-5-R1-1 A6PE.
 4. Themethod of claim 1, wherein the A6PE is a Brevibacillus thermoruber A6PE.5. The method of claim 1, wherein the A6PE is thermostable, the A6PP isthermostable, or both the A6PE and the A6PP are thermostable.
 6. Themethod of claim 2 further comprising converting glucose 6-phosphate(G6P) to the F6P, catalyzed by a phosphoglucoisomerase.
 7. The method ofclaim 6 further comprising converting glucose 1-phosphate (G1P) to theG6P, catalyzed by a phosphoglucomutase.
 8. The method of claim 7 furthercomprising converting a polymeric glucose carbohydrate to the G1P,catalyzed by an α-glucan phosphorylase or a cellodextrin phosphorylase.9. The method of claim 8, wherein the polymeric glucose carbohydrate isselected from starches, cellodextrins, maltodextrins, and glycogens. 10.The method of claim 8 further comprising treating the polymeric glucosecarbohydrate with a debranching enzyme.
 11. The method of claim 10,wherein the debranching enzyme is selected from isoamylases andpullulanases.
 12. The method of claim 3 further comprising convertingG6P to the F6P, catalyzed by a phosphoglucoisomerase.
 13. The method ofclaim 12 further comprising converting G1P to the G6P, catalyzed by aphosphoglucomutase.
 14. The method of claim 13 further comprisingconverting a polymeric glucose carbohydrate to the G1P, catalyzed by anα-glucan phosphorylase or a cellodextrin phosphorylase.
 15. The methodof claim 14, wherein the polymeric glucose carbohydrate is selected fromstarches, cellodextrins, maltodextrins, and glycogens.
 16. The method ofclaim 14 further comprising treating the polymeric glucose carbohydratewith a debranching enzyme.
 17. The method of claim 16, wherein thedebranching enzyme is selected from isoamylases and pullulanases. 18.The method of claim 4 further comprising converting G6P to the F6P,catalyzed by a phosphoglucoisomerase.
 19. The method of claim 18 furthercomprising converting G1P to the G6P, catalyzed by a phosphoglucomutase.20. The method of claim 19 further comprising converting a polymericglucose carbohydrate to the G1P, catalyzed by an α-glucan phosphorylaseor a cellodextrin phosphorylase.
 21. The method of claim 20, wherein thepolymeric glucose carbohydrate is selected from starches, cellodextrins,maltodextrins, and glycogens.
 22. The method of claim 20 furthercomprising treating the polymeric glucose carbohydrate with adebranching enzyme.
 23. The method of claim 22, wherein the debranchingenzyme is selected from isoamylases and pullulanases.
 24. The method ofclaim 1 further comprising producing a cell lysate mixture from cellsthat express the A6PE and cells that express the A6PP.
 25. The method ofclaim 24, wherein the cell lysate mixture comprises a cell lysate fromcells that express both the A6PE and the A6PP, or wherein the celllysate mixture comprises a cell lysate from cells that express A6PE anda cell lysate from cells that express A6PP.
 26. The method of claim 24further comprising incubating the cell lysate mixture with (a) apolymeric glucose carbohydrate, (b) α-glucan phosphorylase or acellodextrin phosphorylase, (c) a phosphoglucomutase, and (d) aphosphoglucoisomerase to produce allulose.
 27. The method of claim 26further comprising incubating the cell lysate mixture with a cell lysatecomprising the α-glucan phosphorylase or the cellodextrin phosphorylase,a cell lysate comprising the phosphoglucomutase, and a cell lysatecomprising the phosphoglucoisomerase.
 28. The method of claim 26,wherein the polymeric glucose carbohydrate is selected from starches,cellodextrins, maltodextrins, and glycogens.
 29. The method of claim 26,wherein the cell lysate mixture further comprises a debranching enzyme.30. The method of claim 29, wherein the debranching enzyme is selectedfrom isoamylases and pullulanases.